Genetically encoded biosensors

ABSTRACT

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of, and claims the benefit of priorityunder 35 U.S.C. § 121 to, U.S. application Ser. No. 16/902,160 filedJun. 15, 2020, now allowed, which is a Continuation of, and claimspriority under 35 U.S.C. § 121 to, U.S. application Ser. No. 16/002,697filed Jun. 7, 2018, which is a Continuation-In-Part of, and claimspriority under 35 U.S.C. § 120 to, U.S. application Ser. No. 15/904,574filed Feb. 26, 2018, which is a Divisional application of, and claimsthe benefit of priority under 35 U.S.C. § 121 to, U.S. application Ser.No. 15/664,326 filed Jul. 31, 2017, which is a Divisional applicationof, and claims the benefit of priority under 35 U.S.C. § 121 to, U.S.application Ser. No. 14/350,199 filed Nov. 18, 2014, which is a U.S.National Phase application of, and claims the benefit of priority under35 U.S.C. 371 to, International Application No. PCT/US2012/059219 filedOct. 8, 2012, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Application No. 61/544,867 filed Oct. 7, 2011.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on 25 Oct. 2021, isnamed 30872_0020003_SEQ.txt, and is 254553 bytes in size.

TECHNICAL FIELD

This disclosure relates to genetically encoded biosensors and methodsfor the design, production, and use of such biosensors.

BACKGROUND

Protein-based sensors that transduce microscopic binding events intomacroscopically observable signals are available to allow real-timevisualization of a variety of biological events and/or molecules(Frommer et al., Chem. Soc. Rev., 38:2833-2841, 2009). Such sensors canbe targeted and/or expressed in living cells, tissues, and organisms,and permit imaging with minimally invasive techniques (Okumoto, Curr.Opin. Biotechnol., 21:45-54, 2010). Application of these sensors islimited by the narrow range of analytes that can be detected and/or bytheir inability to distinguish signal over noise.

SUMMARY

In one aspect, a recombinant peptide biosensor is provided that includesan analyte-binding framework portion and a signaling portion, whereinthe signaling portion is present within the framework portion at a siteor amino acid position that undergoes a conformational change uponinteraction of the framework portion with a defined, specific, orselected analyte.

In one embodiment, the signaling portion is allosterically regulated bythe framework portion such that signaling from the signaling portion isaltered upon interaction of the framework portion with the analyte. Insome embodiments, signaling by the signaling portion detectablyincreases upon interaction of the framework portion with the analyte. Insome embodiments, signaling by the signaling portion detectablydecreases upon interaction of the framework portion with the analyte. Insome embodiments, signaling by the signaling portion is proportional tothe level of interaction between the framework portion and the analyte.

In some embodiments, the signaling portion is a superfolder (SF)fluorescent protein (see, for example, Pedelacq et al., 2006, NatureBiotechnol., 24:79-88), a protein that exhibits robust folding, evenwhen fused to a protein that folds poorly. In some embodiments, the SFprotein is circularly permuted. In some embodiments, the SF protein is agreen fluorescent protein, a yellow fluorescent protein, a redfluorescent protein, or a blue fluorescent protein.

In some embodiments, the framework portion has a first structure in theabsence of an analyte and a second structure, that is detectablydistinct from the first structure, in the presence of the analyte. Insome embodiments, the conformational change between the first structureand the second structure allosterically regulates the signaling portion.In some embodiments, the framework portion is a periplasmic bindingprotein (PBP) or a variant of a PBP.

In some embodiments, the analyte-binding framework portion bindsspecifically to an analyte selected from the group consisting ofglucose, maltose, phosphonate, glutamate, GABA, and ACh.

In another aspect, a recombinant peptide biosensor is provided thatincludes an amino acid sequence having at least 90% identity to arecombinant peptide biosensor selected from the group consisting of SEQID NOs: 176-182, wherein the recombinant peptide biosensor bindsspecifically to glutamate.

In one embodiment, the recombinant peptide biosensor includes arecombinant peptide biosensor selected from the group consisting of SEQID NOs: 176-182 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to glutamate. In some embodiments, the recombinant peptidebiosensor includes a recombinant peptide biosensor selected from thegroup consisting of SEQ ID NOs: 176-182.

In still another aspect, a recombinant peptide biosensor is providedthat includes an amino acid sequence having at least 90% identity to arecombinant peptide biosensor having the sequence shown in SEQ ID NO:183, wherein the recombinant peptide biosensor binds specifically toglucose.

In some embodiments, the recombinant peptide biosensor includes arecombinant peptide biosensor having the sequence shown in SEQ ID NO:183 comprising 10 or fewer conservative amino acid substitutions,wherein the recombinant peptide biosensor binds specifically to glucose.In some embodiments, the recombinant peptide biosensor includes arecombinant peptide biosensor having the sequence shown in SEQ ID NO:183.

In one aspect, a recombinant peptide biosensor is provided that includesan amino acid sequence having at least 90% identity to a recombinantpeptide biosensor having the sequence shown in SEQ ID NO: 184, whereinthe recombinant peptide biosensor binds specifically to GABA.

In one embodiment, the recombinant peptide biosensor includes arecombinant peptide biosensor having the sequence shown in SEQ ID NO:184 comprising 10 or fewer conservative amino acid substitutions,wherein the recombinant peptide biosensor binds specifically to GABA. Inone embodiment, the recombinant peptide biosensor includes a recombinantpeptide biosensor having the sequence shown in SEQ ID NO: 184.

In another aspect, a recombinant peptide biosensor is provided thatincludes an amino acid sequence having at least 90% identity to arecombinant peptide biosensor having a sequence selected from the groupconsisting of SEQ ID NO: 185 and 186, wherein the recombinant peptidebiosensor binds specifically to ACh.

In one embodiment, the recombinant peptide biosensor includes arecombinant peptide biosensor having a sequence selected from the groupconsisting of SEQ ID NO: 185 and 186 comprising 10 or fewer conservativeamino acid substitutions, wherein the recombinant peptide biosensorbinds specifically to ACh. In one embodiment, the recombinant peptidebiosensor includes a recombinant peptide biosensor having a sequenceselected from the group consisting of SEQ ID NO: 185 and 186.

In one aspect, a nucleic acid is provided that encodes a recombinantpeptide biosensor as described herein.

In one aspect, a vector is provided that includes a nucleic acid asdescribed herein.

In one aspect, a cell is provided that includes a nucleic acid asdescribed herein.

In one aspect, a cell is provided that includes a vector as describedherein.

In one aspect, a kit is provided that includes a recombinant peptidebiosensor as described herein, a nucleic acid as described herein, avector as described herein, and/or the cell as described herein.

In still another aspect, a method is provided for detecting glutamate,the method comprising detecting a level of fluorescence emitted by arecombinant peptide biosensor, the peptide biosensor having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 176-182,and correlating the level of fluorescence with the presence ofglutamate.

In some embodiments, the recombinant peptide biosensor is expressed froma nucleic acid. In some embodiments, the method includes contacting therecombinant peptide biosensor with a sample comprising glutamate. Insome embodiments, the method includes correlating the level offluorescence with a concentration of glutamate. In some embodiments, themethod includes comparing the level of fluorescence with a level offluorescence emitted by the recombinant peptide biosensor in thepresence of a sample comprising a known concentration or range ofconcentrations of glutamate. In some embodiments, the method isperformed in vitro.

In some aspects, a method for detecting glucose is provided, the methodcomprising detecting a level of fluorescence emitted by a recombinantpeptide biosensor, the peptide biosensor having an amino acid sequenceshown in SEQ ID NO: 183, and correlating the level of fluorescence withthe presence of glucose.

In some aspects, a method for detecting GABA is provided, the methodcomprising detecting a level of fluorescence emitted by a recombinantpeptide biosensor, the peptide biosensor having an amino acid sequenceshown in SEQ ID NO: 184, and correlating the level of fluorescence withthe presence of GABA.

In some aspects, a method for detecting ACh is provided, the methodcomprising detecting a level of fluorescence emitted by a recombinantpeptide biosensor, the peptide biosensor having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 185 and 186, andcorrelating the level of fluorescence with the presence of ACh.

In some aspects, a method for detecting a defined, selected, or specificanalyte is provided, the method comprising detecting a level offluorescence emitted by a recombinant peptide biosensor of claim 1; andcorrelating the level of fluorescence with the presence of a defined,selected, or specific analyte

In some embodiments, the recombinant peptide biosensor is expressed froma nucleic acid. In some embodiments, the method includes contacting therecombinant peptide biosensor with a sample comprising the analyte. Insome embodiments, the method includes correlating the level offluorescence with a concentration of the analyte. In some embodiments,the method includes comparing the level of fluorescence with a level offluorescence emitted by the recombinant peptide biosensor in thepresence of a sample comprising a known concentration or range ofconcentrations of the analyte. In some embodiments, the method isperformed in vitro. In some embodiments, the analyte is selected fromthe group consisting of glutamate, glucose, GABA, and ACh.

The present disclosure provides genetically encoded recombinant peptidescontaining an analyte-binding framework portion linked (e.g., operablylinked) to a signaling portion, wherein the signaling portion isallosterically regulated by the framework portion upon interaction ofthe framework portion with an analyte (e.g., a defined, selected, and/orspecific analyte). These constructs can be used as biosensors, e.g., totransduce microscopic binding events into macroscopically observablesignals.

The present disclosure provides, in part, recombinant peptides for useas biosensors (e.g., recombinant peptide biosensors) that include (e.g.,comprise, consist essentially of, or consist of), e.g., include atleast, an analyte-binding framework portion and a signaling portion. Asdescribed in further detail herein, such signaling portions are presentwithin the framework portion at a site or amino acid position thatundergoes a conformational change (e.g., a conformational changesufficient to alter a physical and/or functional characteristic of thesignaling portion, e.g., a substantial conformational change) uponinteraction of the framework portion with a defined, specific, orselected analyte (e.g. such as an analyte to which the framework portionor a region thereof, and/or the biosensor, specifically binds).

For example, in some instances, the signaling portion is allostericallyregulated by the framework portion such that signaling from thesignaling portion is altered (e.g. wherein a first level of signaling isaltered or changed to a second level of signaling that can bedistinguished using routine methods of detection from the first) uponinteraction of the framework portion with the analyte. In someinstances, signaling by the signaling portion can detectably increase ordecrease upon interaction of the framework portion with the analyte. Insome instances, signaling by the signaling portion upon interaction ofthe biosensor with a defined, specific, or selected analyte (e.g. suchas an analyte to which the framework portion or a region thereof, and/orthe biosensor, specifically binds) can be proportional or can correlatewith to the level of interaction between the framework portion and theanalyte such that the level of interaction can be determined from thesignaling or alteration thereof.

In some instances, framework portions of the biosensors disclosed hereinhave a first structure in the absence of an analyte and a secondstructure that is detectably distinct from the first structure in thepresence of the analyte. In some instances, the conformational changebetween the first structure and the second structure allostericallyregulates the signaling portion.

In some instances, framework portions of the biosensors disclosed hereincan be, or can include (e.g., comprise, consist essentially of, orconsist of), periplasmic binding proteins (PBP) or variants of a PBP. Insome instances, exemplary PBPs or variants thereof can include, but arenot limited to, peptides with at least 90% identity to a peptideselected from the group consisting of SEQ ID NO:105, SEQ ID NO: 106, SEQID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO:111,SEQ ID NO:113, and SEQ ID NO:114. In some instances, exemplary PBPs orvariants thereof can include, but are not limited to, peptides with atleast 95% identity to a peptide selected from the group consisting ofSEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ IDNO:109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114.In some instances, exemplary PBPs or variants thereof can include, butare not limited to, peptides selected from the group consisting of SEQID NO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114. In someinstances, exemplary PBPs or variants thereof can include, but are notlimited to, peptides selected from the group consisting of SEQ IDNO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114 comprising10 or fewer conservative amino acid substitutions. PBPs or variantsthereof disclosed herein can be truncated.

In some instances, signaling portions of the biosensors disclosed hereincan be or can include (e.g., comprise, consist essentially of, orconsist of) one or more (e.g., one, two three, four, five, and less thanten) circularly permuted fluorescent proteins (cpFPs). Such cpFPs can beinclude but are not limited to, for example, green fluorescent proteins,yellow fluorescent proteins, red fluorescent proteins, and/or bluefluorescent proteins.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to glucose. Such sensors can be referred to as glucosebinding biosensors or glucose biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to maltose. Such sensors can be referred to as maltosebinding biosensors or maltose biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to phosphonate.

Such sensors can be referred to as phosphonate binding biosensors orphosphonate biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to glutamate. Such sensors can be referred to as glutamatebinding biosensors or glutamte biosensors.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, and 53, wherein the recombinant peptide biosensor binds specificallyto maltose; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53comprising 10 or fewer conservative amino acid substitutions, whereinthe recombinant peptide biosensor binds specifically to maltose; and/ora recombinant peptide biosensor selected from the group consisting ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 62 and 63, wherein therecombinant peptide biosensor binds specifically to glutamate; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 62 and 63 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to glutamate; and/or a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 62 and 63.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 77 and 78, wherein therecombinant peptide biosensor binds specifically to phosphonate; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 77 and 78 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to phosphonate; and/or a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 77 and 78.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 91, 92, 93 and 94, wherein therecombinant peptide biosensor binds specifically to glucose; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 91, 92, 93 and 94 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to glucose; and/or a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 91, 92, 93 and 94.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): SEQ ID NO:91; SEQ IDNO:92; SEQ ID NO:93; SEQ ID NO:95.

In some instances, any recombinant biosensor disclosed herein can beisolated and/or purified. The terms “isolated” or “purified,” whenapplied to a biosensor disclosed herein includes nucleic acid proteinsand peptides that are substantially free or free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free or free of precursors or other chemicals whenchemically synthesized.

The disclosure also provides, in part, nucleic acids (e.g., isolatedand/or purified nucleic acids) encoding any one or more of therecombinant peptide biosensors disclosed herein. For example, nucleicacids can encode: an amino acid sequence with at least 90% identity to arecombinant peptide biosensor selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53, wherein therecombinant peptide biosensor binds specifically to maltose; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to maltose; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, and 53; an amino acid sequence with at least 90% identity toa recombinant peptide biosensor selected from the group consisting ofSEQ ID NO: 62 and 63, wherein the recombinant peptide biosensor bindsspecifically to glutamate; a recombinant peptide biosensor selected fromthe group consisting of SEQ ID NO: 62 and 63 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to glutamate; a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 62 and 63; anamino acid sequence with at least 90% identity to a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 77 and 78,wherein the recombinant peptide biosensor binds specifically tophosphonate; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 77 and 78 comprising 10 or fewer conservativeamino acid substitutions, wherein the recombinant peptide biosensorbinds specifically to phosphonate; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 77 and 78; an aminoacid sequence with at least 90% identity to a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 91, 92, 93and 94, wherein the recombinant peptide biosensor binds specifically toglucose; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 91, 92, 93 and 94 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to glucose; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 91, 92, 93 and 94;and/or SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:95.

In some instances, the disclosure includes vectors containing one or aplurality of the nucleic acids disclosed herein and cells containingsuch vectors. In some instances, the disclosure provides cellscontaining one or a plurality of nucleic acids disclosed herein.

In some instances, the disclosure includes kits related to thebiosensors and nucleic acids disclosed herein Such kits can include orcontain, for example, a biosensor, a nucleic acid encoding a biosensor,vectors, and/or cells, provided herein.

In some instances, the disclosure provides methods related to thebiosensors and nucleic acids disclosed herein. Such methods can includemethods of making, using, and/or selling the biosensors and nucleicacids disclosed herein. For example, methods can include methods forproducing genetically encoded recombinant peptide biosensors. In suchinstances, methods can include, for example, selecting a frameworkportion that binds specifically to a target analyte and that undergoes aconformational change upon interacting binding to the target analyte,identifying a site or amino acid position within the selected frameworkportion where or around which the conformational change occurs, andinserting a signaling portion into the site or amino acid position. Insome instances, framework portions include periplasmic binding proteins(PBPs) disclosed herein. Exemplary PBPs include PBPs that bind (e.g.,bind specifically) to glucose.

In some instances, the present disclosure includes methods for detectingglucose, e.g., in a sample containing a level of glucose. Such methodscan include, detecting a level of fluorescence emitted by a recombinantpeptide biosensor, the peptide biosensor having an amino acid sequenceselected from the group consisting of SEQ ID NO: 91, 92, 93 and 94, andcorrelating the level of fluorescence with the presence of glucose. Insome instances, recombinant peptide biosensors used in the methodsherein are expressed from nucleic acids. In some instances, methodsinclude contacting the recombinant peptide biosensor with a test sample(e.g., a sample comprising glucose). In some instances, methods caninclude the level of fluorescence emitted by a biosensor (e.g., abiosensor bound to glucose) with a concentration glucose in the sample.Such correlation can include, for example, comparing the level offluorescence with a level of fluorescence emitted by the recombinantpeptide biosensor in the presence of a sample comprising a knownconcentration or range of concentrations of glucose. In some instance,the level of fluorescence emitted by the recombinant peptide biosensorin the presence (e.g., bound or bound specifically to) of a samplecomprising a known concentration or range of concentrations of glucoseis stored on an electronic database.

One of skill will appreciate that such methods can be adapted for anydefined, specific, or selected analyte. For example, in some instances,the disclosure provides methods for detecting a defined, selected, orspecific analyte. These methods can include detecting a level offluorescence emitted by a recombinant peptide biosensor expressed from anucleic acid and correlating the level of fluorescence with the presencethe defined, selected, or specific analyte. In some instances, methodsinclude contacting the recombinant peptide biosensor with a samplecomprising the analyte. In some instances, methods include correlatingthe level of fluorescence with a concentration of the analyte. In someinstances, methods include comparing the level of fluorescence with alevel of fluorescence emitted by the recombinant peptide biosensor inthe presence of a sample comprising a known concentration or range ofconcentrations of the analyte, wherein the level of fluorescence emittedby the recombinant peptide biosensor in the presence of a samplecomprising a known concentration or range of concentrations of theanalyte is stored on an electronic database.

In some instances, the present disclosure provides methods for detectinga defined, selected, or specific analyte, the method comprisingdetecting a level of fluorescence emitted by a recombinant peptidebiosensor of any one of claims 1-36; and correlating the level offluorescence with the presence of a defined, selected, or specificanalyte. In some instances, recombinant peptide biosensors can beexpressed from a nucleic acid. In some instances, methods can includecontacting the recombinant peptide biosensor with a sample comprisingthe analyte. In some instances, methods can include correlating thelevel of fluorescence with a concentration of the analyte and,optionally, comparing the level of fluorescence with a level offluorescence emitted by the recombinant peptide biosensor in thepresence of a sample comprising a known concentration or range ofconcentrations of the analyte. In some instances, the level offluorescence emitted by the recombinant peptide biosensor in thepresence of a sample comprising a known concentration or range ofconcentrations of the analyte is stored on an electronic database.

Methods herein can be performed in vitro.

In some instances, the present disclosure provides compositionscontaining any one or a plurality of the peptide biosensors and/ornucleic acids disclosed herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 Cartoon representation showing ligand bound Escherichia colimalto-dextrin-binding protein (EcMBP) and potential circularly-permutedfluorescent protein (cpFP) insertion sites.

FIG. 2 Cartoon representation showing ligand bound Pyrococcus furiosusmaltotriose binding protein (PfMBP) and potential cpFP insertion sites.

FIG. 3 Cartoon representation showing ligand bound E. coliglutamate-binding protein (EcYbeJ) and potential cpFP insertion sites.

FIG. 4 Cartoon representation showing ligand bound E. coliphosphonate-binding protein (EcPhnD) and potential cpFP insertion sites.

FIG. 5 Cartoon representation showing ligand bound Thermus thermophilusglucose binding protein (TtGBP) and potential cpFP insertion sites.

FIG. 6A-B Changes in EcMBP upon maltose binding and locations at whichcircularly-permuted fluorescent protein (cpFP) was inserted are shown ascolored spheres at the Cα positions. Yellow: 165-166, Green: 175-176,Cyan: 311-312, Violet: 317-318 (FIG. 6A). (FIG. 6B) shows backbonestructural changes. The Cα dihedral is calculated from the four atoms:Cαi+2, Cαi+1, Cαi, Cαi−1. ΔDihedral is calculated as the difference indihedrals between the closed (1ANF) and open (1OMP) states of MBP, andcorrected to fall within a range of −180° to 180°. The regions nearresidues 175 and 311 are labeled. There is a crystallographic artifactat the N-terminus resulting in the appearance of significant structuralchanges.

FIG. 7A Amino acid sequence of MBP-165-cpGFP (SEQ ID NO:1).

FIG. 7B Amino acid sequence of MBP-165-cpGFP.PPYF (SEQ ID NO:2).

FIG. 7C Amino acid sequence of MBP-165-cpGFP.PCF (SEQ ID NO:3).

FIG. 8A Amino acid sequence of MBP-175-cpGFP (SEQ ID NO:4).

FIG. 8B Amino acid sequence of MBP-175-cpGFP.L1-HL (SEQ ID NO:5).

FIG. 9A Amino acid sequence of MBP-311-cpGFP (SEQ ID NO:6).

FIG. 9B Amino acid sequence of MBP-311-cpGFP.L2-NP (SEQ ID NO:7).

FIG. 10 Amino acid sequence of MBP-317-cpGFP (SEQ ID NO:8).

FIGS. 11A-11D Line charts showing EcMBP plot of ΔF/F for clarifiedlysate screen of cpGFP linker-screens at insertion points 165, 175, 311,and 317. The horizontal dashed line at zero indicates no fluorescencechange. Standard deviations in ΔF/F are less than 10% of an average ΔF(repetitions for MBP165-cpGFP.PPYF yields ΔF/F values of 2.51, 2.63, and2.54).

FIG. 12 Isothermal titration calorimetry (ITC) of MBP317-cpGFP withmaltose.

FIG. 13 Graph showing EcMBP165-cpGFP.PPYF affinity variant bindingmaltose-binding curves. Binding curves for affinity variants ofMBP165-cpGFP.PPYF. Data is fit to a single-binding site isotherm.Curve-fit affinities are: WT binding pocket, 5 μM (●); W230A, 32 μM (▪);W62A, 375 μM (▴); W340A, >1 mM (▾); 1329W, 11 μM (□).

FIGS. 14A-14D Line graphs showing maltose and sucrose binding curves forwild-type and 5-7 variants of the EcMBP-cpGFP sensors. Maltose (black)and sucrose (red) binding curves for wild-type (filled, solid lines) and5-7 variants (open, dashed lines) of the MBP-cpGFP sensors.MBP165-cpGFP.PPYF (FIG. 14A); MBP165-cpGFP.PCF (FIG. 14B);MBP175-cpGFP.L1-HL (FIG. 14C); MBP311-cpGFP.L2-NP (FIG. 14D).

FIGS. 15A-15D Line graphs showing emission spectra for colored variantsof EcMBP sensors. Fluorescence emission spectra of the MBP165-Blue,Cyan, Green, and Yellow wild-type sensors (FIG. 15A) and the 5-7variants (FIG. 15B) in the absence of ligand (dashed lines, opencircles), with 10 mM maltose (solid lines, filled circles), or 10 mMsucrose (solid lines, filed squares). Sensors were excited at 383, 433,485, and 485 nm, respectively. Titration of maltose and sucrose in theBlue, Cyan, Green, and Yellow MBP165 wild-type sensors (FIG. 15C) andfor the 5-7 variants (FIG. 15D). Filled circles are titration ofmaltose, open circles are titration of sucrose. For the wild-typesensors, Kds for maltose binding are: Blue 3.3 Cyan 13 μM, Green 4.5 μM,Yellow 3.3 μM. No sucrose binding is observed. For the 5-7 variants, Kdof Green is 2.4 mM (sucrose) and 7.1 mM (maltose). Kd of Yellow is 2.5mM (sucrose) and 4.5 mM (maltose).

FIG. 16 Plot of ΔF/F for clarified lysate screen of MBP165-cpBFPlinker-screen. The horizontal dashed line at zero indicates nofluorescence change.

FIGS. 17A-17B Line graphs showing maltose binding. Blue (wt bindingpocket) has an affinity of 2.7 μM. Green (W230A) has an affinity of 40μM. Yellow (W62A) has an affinity of 350 μM. Cyan (W340A) has anaffinity of approximately 1.7 mM. Data is plotted at ΔF/F (FIG. 17A) ornormalized to Fractional Saturation (FIG. 17B).

FIGS. 18A-18C Images bacterial cells expressing (FIG. 18A) EGFP, (FIG.18B) PPYF, or (FIG. 18C) PPYF.T203V in the absence (top) and presence(bottom) of maltose.

FIGS. 19A-19B Line graphs showing EcMBP-cpGFP.PPYF.T203V 2-photonexcitation spectra. MBP165-cpAzurite.L2-FE (FIG. 19A), -cpCFP.PCF (FIG.19A), -cpGFP.PPYF (FIG. 19B), and -cpYFP.PPYF (FIG. 19B) were excited atthe wavelengths indicated and emission measured through appropriatewavelength filters. Two graphs are shown to present different y-axisscales. Optimal ΔF/F values for 2-photon excitation of the spectralvariants of MBP165 are: −cpAzurite, 1.1 (ex 760 nm); −cpCFP, 2.3 (ex830-960 nm); −cpGFP, 10.0 (ex 940 nm); −cpYFP, 2.6 (ex 940 nm).

FIGS. 20A-20C Images showing EcMBP-cpGFP.PPYF.T203V expressing HEKcells. Images of individual HEK293 cells expressing membrane displayedPPYF.T203V in the absence of maltose (FIG. 20A), in the presence of 1 mMmaltose (FIG. 20B), and after washout with maltose-free buffer (FIG.20C). Scale bars are 10 μm.

FIGS. 21A-21B Graphs showing quantification of fluorescence ofEcMBP-cpGFP.PPYF.T203V when displayed on the surface of HEK cells. (FIG.21A) Concentration dependence. (FIG. 21B) Observed fluorescence after a“puff” of HBSS solution containing 1 mM maltose and 2.5 nM Alexa Fluor®568 (Invitrogen, Carlsbad, Calif.).

FIGS. 22A-22D Cartoon representations and close-up views of inter-domainlinkers and selected amino acids of the cpGFP chromophore environment ofthe structure of MBP175-cpGFP.L1-HL (FIG. 22A and FIG. 22B) andMBP311-cpGFP.L2-NP (FIG. 22C and FIG. 22D) bound to maltose. The MBPdomain is colored as in FIG. 1. The cpGFP domain is green and theinter-domain linkers are colored white. The cpGFP chromophore isdisplayed as sticks and the bound maltose as red and white spheres.Ordered water molecules are represented as red spheres. Selectedhydrogen bonds are displayed as dashed black lines. β-strands 10 and 11of cpGFP are displayed as semi-transparent for clarity. The 2Fo-Fcelectron density map calculated with the displayed residues omitted fromthe model is shown as blue mesh.

FIGS. 23A-23D EcMBP-cpGFP: effect of T203V mutation on fluorescence.(FIG. 23A) Emission spectra of 1 μM purified eGFP (filled circles),cpGFP (filled squares), MBP165-cpGFP.PPYF (open circles), andMBP165-cpGFP.PPYF+T203V (open squares) in the absence (dashed lines) orpresence (solid lines) of 1 mM maltose. cpGFP is half as bright as eGFP,and the saturated MBP165-cpGFP.PPYF variants are about half as bright ascpGFP. (FIG. 23B) Titration of maltose for MBP165-cpGFP.PPYF (filledsquares), and MBP165-cpGFP.PPYF+T203V (filled circles). Affinities foreach protein are the same, but with different ΔF/F. (FIG. 23C) Emissionspectra of 1 μM purified eGFP (filled circles), cpGFP (filled squares),MBP311-cpGFP.L2-NP (open circles), and MBP311-cpGFP.L2-NP+T203V (opensquares) in the absence (dashed lines) or presence (solid lines) of 1 mMmaltose. Note that mutation T203V decreases the fluorescence of both theapo-state and the saturated state of MBP311-cpGFP.L2-NP. (FIG. 23D)Titration of maltose for MBP311-cpGFP.L2-NP (filled squares), andMBP311-cpGFP.L2-NP+T203V (filled circles). Affinities for each proteinare the same, but with ΔF/F slightly increased for the T203V variant.

FIG. 24A Amino acid sequence of PfMBP171-cpGFP (SEQ ID NO:50)

FIG. 24B Amino acid sequence of PfMBP171cpGFP.L2-FE (SEQ ID NO:51)

FIG. 25A Amino acid sequence of PfMBP316-cpGFP (SEQ ID NO:52)

FIG. 25B Amino acid sequence of PfMBP316-cpGFP.L1-NP (SEQ ID NO:53)

FIG. 26A-26B Plot of ΔF/F for clarified lysate screen of cpGFPlinker-screens at insertion points 171 (FIG. 26A) and 316 (FIG. 26B).

FIGS. 27A-27D Plot of Beta-sheet circular dichroism (CD) signal as afunction of temperature.

FIGS. 28A-28B PfMBP Fluorescence vs. temperature. (FIG. 28A) Plot offluorescence as a function of temperature in the presence (solid) orabsence (dashed) of ligand. (FIG. 28B) Plot of ΔF/F as a function oftemperature. Using the data from FIG. 27A, ΔF/F for each protein(Fbound-Fapo/Fapo) was calculated for each temperature.

FIGS. 28C-28E Line graphs showing the function of immobilized andsoluble proteins.

FIG. 29A Amino acid sequence of EcYbeJ253-cpGFP (SEQ ID NO:62).

FIG. 29B Amino acid sequence of EcYbeJ253-cpGFP.L1LVL2NP (SEQ ID NO:63).

FIG. 30 EcYbeJ binding curves. Plot of ΔF/F as a function of[Glutamate], μM. The first generation sensor, EcYbeJ253.L1-LV (with theA184V) mutation (grey, solid) has an affinity for glutamate of about 100μM and a ΔF/F of 1.2. The reversion of that affinity mutation, V184A, inthe L1-LV background increases affinity to 1 (grey dashed). The secondgeneration sensor, with the L2-NP linker optimization and the A184Vmutation, has a ΔF/F of at least 4 and an affinity for glutamate ofabout 100 μM (black solid).

FIG. 31 EcYbeJ Hema/cMyc analysis. The effect of N- and C-terminal tagson ΔF/F and glutamate affinity were determined by expressing variouslytagged versions of the EcYbeJ253.L1LVL2NP protein in bacteria. Thepresence of the pRSET leader sequence (black) has no effect on ΔF/F (˜5)or affinity (˜120 when compared to the version without a tag (grey). Theaddition of the cMyc tag to the C-terminus retains ΔF/F and increasesaffinity slightly, to 60 μM. The addition of the N-terminalhemagglutinin tag, with (green) or without (orange) the cMyc tag,decreases ΔF/F substantially.

FIGS. 32A-32B EcYbeJ253-cpGFP.L1LVL2NP.pMinDis expressed in HEK293cells. (FIG. 32A) Images of the sensor expressing HEK cells in theabsence of glutamate (left), with 100 μM glutamate (center), andre-imaged after wash-out of glutamate with buffer (right). (FIG. 32B) Bymeasuring the equilibrium ΔF/F with different concentrations ofglutamate in the buffer, an in situ binding affinity (black) can beobtained. The surface displayed sensor has a higher affinity (3 μM) forglutamate than the soluble sensor (grey), which is about 90 μM.

FIG. 33 EcYbeJ253-cpGFP.L1LVL2NP.pMinDis expressed in neuronal culture,and responds rapidly to added glutamate (green). Red shows signal of 2.5nM Alexa Fluor® 568 (Invitrogen, Carlsbad, Calif.), also in pipette.

FIG. 34A Amino acid sequence of EcPhnD90-cpGFP (SEQ ID NO:77).

FIG. 34B Amino acid sequence of EcPhnD90-cpGFP.L1AD+L297R+L301R (SEQ IDNO: 78).

FIGS. 35A-35C EcPhnD90-cpGFP Binding Curves. For both the L1AD and theL1AD+L297R+L301R variants, binding was determined for (FIG. 35A)2-aminoethylphosphonate (2AEP), (FIG. 35B) methylphosphonate (MP), and(FIG. 35C) ethylphosphonate (EP).

FIGS. 36A-36C The crystal structures of the ligand-free (FIG. 36A), openstate (with H157A mutation to the binding pocket) and the ligand-bound(FIG. 36B), closed state of EcPhnD clearly shows a large conformationalchange. Residues in between which cpGFP is inserted in EcPhnD90-cpGFPare marked by red spheres, in the equatorial strand (red). (FIG. 36C)Analysis of the change in Cα dihedral (ΔDihedral) clearly shows thatresidues for which there is the greatest ΔDihedral upon going from theopen to the closed state are residues 88 (ΔDihedral=)−75°, 89(ΔDihedral=123°), and 90 (ΔDihedral=52°).

FIG. 37A Amino acid sequence of TtGBP326-cpGFP (SEQ ID NO:91).

FIG. 37B Amino acid sequence of TtGBP326.L1-PA (SEQ ID NO:92).

FIG. 37C Amino acid sequence of TtGBP326.H66A (SEQ ID NO:93).

FIG. 37D Amino acid sequence of TtGBP326.H348A (SEQ ID NO:94).

FIG. 38 TtGBP326-cpGFP Binding Curves. Plot of ΔF/F as a function of[Glucose], mM.

FIG. 39 An image showing TtGBP326-cpGFP expressed as a transgenicreporter of intracellular glucose in cultured human cells.

FIGS. 40A-40B Are line graphs showing that the addition of extracellularglucose increases TtGBP326-cpGFP fluorescence in human cells.

FIG. 41 Amino acid sequence of Escherichia coli maltodextrin-bindingprotein (EcMBP) (SEQ ID NO: 105).

FIG. 42 Amino acid sequence of Pyrococcus furiosus maltose-bindingprotein (PfMBP) (SEQ ID NO: 106).

FIG. 43 Amino acid sequence of E. coli glutamate-binding protein(EcYbeJ) (SEQ ID NO:107).

FIG. 44 Amino acid sequence of E. coli phosphonate-binding protein(EcPhnD) (SEQ ID NO:108).

FIG. 45 Amino acid sequence of Thermus thermophilus glucose-bindingprotein (TtGBP) (SEQ ID NO:109).

FIG. 46 Amino acid sequence of UniProt accession number Q92N37 (SEQ IDNO: 110).

FIG. 47 Amino acid sequence of UniProt accession number DOVWX8 (SEQ IDNO:111).

FIG. 48 Amino acid sequence of UniProt accession number Q7CX36 (SEQ IDNO:112).

FIG. 49 Amino acid sequence of UniProt accession number POAD96 (SEQ IDNO:113).

FIG. 50 Amino acid sequence of TtGBP326.L1PA.L2NP.H66A.H348A.L276V (SEQID NO:114).

FIG. 51 A line graph showing binding ofTtGBP326.L1PA.L2NP.H66A.H348A.L276V to glucose.

FIG. 52 A line graph showing fluorescence increase upon addition ofglucose to HEK293 cells expressing TtGBP326.LIPA.L2NP.H66A.H348A.L276Von their extracellular surface.

FIG. 53A-D SF-iGluSnFR.A184S shows larger responses to visual stimulithan SF-iGluSnFR.A184V. (FIG. 53A) Two-photon standard-deviationprojection of SF-iGluSnFR.A184S and A184V expressed in ferret visualcortex (A184S: 190 μm, A184V: 175 μm, scale bar 100 μm). (FIG. 53B)Trial-averaged stimulus-evoked responses (shown for ROI 1) reveal robustorientation tuning and peak amplitudes of ˜30% ΔF/F for A184S. Peakresponses plotted as a function of stimulus orientation show robustselectivity with the A184S variant. For the A184V variant,stimulus-evoked fluctuations are too small (˜5% ΔF/F) to generate robusttuning plots. (FIG. 53C) Two-photon standard-deviation projection of anisolated dendritic segment with active spines revealed withSF-iGluSnFR.A184S. Individual dendritic spines are driven selectivelyand strongly by drifting gratings. Orientation tuning from peakresponses shows large spine responses (30-50% ΔF/F) and, importantly,reveals that spines on a single dendritic branch can receive differentlytuned excitatory input. (FIG. 53D) Same as in (FIG. 53C) forSF-iGluSnFR.A184V. Dendritic spine responses with A184V are weak andalmost unresolvable.

FIG. 54A-D SF-iGluSnFR. S72A permits resolution of multiple glutamaterelease events in cultured mouse embryonic hippocampal neurons. (FIG.54A) Single (dashed) and averaged (solid) traces of SF-iGluSnFR.S72A(blue) and SF-iGluSnFR.A184V (red) response to 20 Hz paired electricalstimuli. (FIG. 54B) Histogram showing intensity second pulse to firstpulse response. (FIG. 54C) The faster off-rate of S72A can be used toobserve vesicle release depression. Higher concentrations ofextracellular calcium can increase vesicle release, leading to vesicleexhaustion as the train of field pulses progresses. (FIG. 54D) The slowdecay of A184V obscures this depression.

FIG. 55A-D S72A variant shows faster bouton fluorescence signalsresulting from single or trains of electrical stimulation mousecerebellar brain slice. (FIG. 55A) Averaged response from single boutonsexpressing GCaMP6f (GC6f) at 2 mM [Ca²⁺]_(extracellular) (green), GC6fat 1.5 mM [Ca²⁺]_(e) (black), SF-iGluSnFR.A184V at 1.5 mM [Ca²⁺]_(e)(A184V, red) and SF-iGluSnFR.S72A at 1.5 mM [Ca²⁺]_(e) (S72A, blue),normalized to peak response. In parenthesis the number of trials used tocalculate the average. Right, summary plots of ΔF/F₀, 10-90% rise time,50% decay time and signal-to-noise-ratio (SNR). Multiple comparisonswere performed with the Kruskal-Wallis test and the Dunn's multiplecomparisons test. *P<0.05, **P<0.01, ****P<0.0001. (FIG. 55B) Two-photonfluorescent image of granule cells and parallel fibers expressing A184Vin cerebellum slice (GL—granule layer, ML—molecular layer). Yellowarrows indicate labeled soma of granule cells, and circle indicateboutons from parallel fibers. Bottom, example of single trial A184Vfluorescence responses to 20 Hz electrical stimulation (red) and theaverage of 10 trials (purple). (FIG. 55C) Population averagefluorescence responses to 20 Hz stimulation (n boutons=5 GC6f; n=17,A184V; n=3, S72A). Traces are normalized to the peak of the firstresponse. (FIG. 55D) Population average of response to 100 Hz electricalstimulation (n boutons=9 GC6f; n=9, A184V; n=7, S72A) normalized to themaximum amplitude (left) or to the peak of the first response (middle),and average response of all the boutons. n is number of boutons. Blackarrows indicate time of electrical stimulation.

FIG. 56A-B High-speed two photon imaging (1016 Hz frame rate) of aneuron expressing SF-Venus-iGluSnFR. (FIG. 56A) RuBi-glutamate wasuncaged for 10 msec. at each of two 5 μm spots (red arrowheads) on thedendrites. Saturation denotes the glutamate transient amplitude. Yellowline indicates locations for traces shown in (FIG. 56B). (FIG. 56B)Recorded traces at nine pixels at various distances from the uncagingfocus, along the yellow line in (FIG. 56A). The traces are approximatemaximum likelihood solutions recovered with the FADE algorithm.(Kazemipour et al., Proceedings of the 2017 Asilomar Conference onSignals, Systems, and Computers, October 29-November 1, Pacific Grove,Calif.), which incorporates dynamics having arbitrarily fast rise butslow decay. This recording is of a single uncaging event, withoutaveraging.

FIG. 57 Circular dichroism of iGluSnFR and SF-iGluSnFR. 20 μM purifiedand dialyzed protein in 0.1×PBS was analyzed by circular dichroism(Chirascan, Applied Biophysics). Grey, iGluSnFR; black, SF-iGluSnFR;green, cpSFGFP; thick line, with 1 mM glutamate; thin line, noglutamate. Spectra were collected with a 1 sec. sampling time afterequilibration for 2 min at each temperature. The first unfoldingtransition is shifted from about 50° C. to 55° C. by inclusion of theSuperfolder mutations to cpGFP. Interestingly, the second transition, atabout 75° C., which parallels the transition of cpSFGFP alone, isunchanged.

FIG. 58A-D Spectra of SF-iGluSnFR. (FIG. 58A) 2-photon cross-section ofpurified, soluble iGluSnFR (grey) and SF-iGluSnFR (black) in theligand-free (dashed line) and glutamate-saturated (solid line) state.Excitation (FIG. 58B), emission (FIG. 58C), and absorption spectra (FIG.58D) of iGluSnFR (grey), SF-iGluSnFR (black), and cpSFGFP (green) withglutamate (solid line) and without (dashed line).

FIG. 59A-E Representative images of (FIG. 59A) SF-iGluSnFR and (FIG.59B) iGluSnFR in mouse somatosensory cortex taken at 0.9 μm/pixel, 0.126nsec dwell time per μm, 80 mW power, prior to bleaching. 20 nl ofAAV2/1.hSynapsin1.iGluSnFR or SF-iGluSnFR (identical virus titer,prepared by the same person) was injected three weeks before imaging.Contrast adjusted to 10 grayscales in both images to make originaliGluSnFR observable. Mean signal-to-noise ratios (n=2 animals) are 66vs. 14 (80 mW power) and 2.4 vs. 0.3 (5 mW power). (FIG. 59C) & (FIG.59D) Representative images of SF-iGluSnFR and iGluSnFR taken with 5 mWpower, which is more typical in live imaging conditions. (FIG. 59E)Bleaching of SF-iGluSnFR (black) and original iGluSnFR (grey) at 80 mWpower and 10× zoom (0.09 μm/pixel, 1.26 nsec dwell time per μm).

FIG. 60A-B In vitro binding affinity. (FIG. 60A) Titration ofbacterially expressed iGluSnFR and SF-iGluSnFR and variants. Affinities(K_(d)) for original iGluSnFR, SF-iGluSnFR.A184S, SF-iGluSnFR.A184V, andSF-iGluSnFR.S72A are 84±7 μM, 7.5±0.4 μM, 41±7 μM, and 200±5 μMrespectively. (FIG. 60B) Kinetics of glutamate binding by stopped-flowfluorescence spectroscopy. Equal volumes of 1 μM SF-iGluSnFR (A184S,A184V, or S72A) and glutamate (variable concentration) were mixed in anSX.18MV stopped-flow spectrometer (Applied Photophysics, Surrey, UK).Representative traces shown. Pseudo-first order analysis indicates thatthe on-rate of binding for SF-iGluSnFR.A184S, A184V, S72A are 6, 5, and0.6 μM⁻¹ sec⁻¹, respectively. The off rates, as determined by they-intercept, are 25 sec⁻¹, 52 sec⁻¹, and 108 sec⁻¹ respectively. Errorbars are standard deviation of three measurements.

FIG. 61 Binding affinity screening. Pellets of bacterially expressedA184X variants of iGluSnFR were washed 5 times in PBS to remove boundglutamate. After freezing and thawing, pellets were clarified bycentrifugation and titrated with glutamate to screen for their affinityfor glutamate. There is a general trend of larger amino acids resultingin weaker affinity.

FIG. 62 Affinity of SF-iGluSnFR variants displayed on the surface ofneurons. AAV2/1.hSynapsin1.SF-iGluSnFR variants (1 μl of 1E13 GC/ml)were used to infect rat hippocampal neuronal culture 3 days afterculturing. After 10 days in vitro, fluorescence was monitored undercontinuous flow of buffer with varying concentrations of glutamate.Affinities (K_(d)) for SF-iGluSnFR.A184S, SF-iGluSnFR.A184V, andSF-iGluSnFR.S72A are 0.6, 2.1, and 34 μM respectively. Affinities forSF-Venus.A184V and SF-Azurite.A184V are 2.0 and 9 μM respectively.Bottom panel is zoom-in of top panel.

FIG. 63A-B Rise and decay of fluorescence signal resulting from a singlefield stimulation (1 msec., 90 mA) in rat hippocampal culture (10 DIV, 7DPI) in non-flowing buffer (FIG. 63B). Traces in FIG. 63A are theaverage of three ROIs (bottom) and three trials (top); error bars arestandard deviation of those nine measurements. The large error forGCaMP6f results from back propagating action potentials, which can beseen in differences from individual ROIs.

FIG. 64 Examples of individual responses for ROIs 1, 2, and 3 (top).Responses of individual Spines #2 and #3 (from FIG. 53) (middle).Histogram showing distribution of spine responses (bottom). Responseamplitudes across individual trials were consistently greater for A184Sthan the A184V when examining all stimulus-evoked responses.

FIG. 65A-D Mouse neuronal culture images. The fluorescent labelingpattern and intensity of primary hippocampal neurons transduced withAAV2/1.hSynapsin1. SF-iGluSnFR.S72A or withAAV2/1.hSynapsin1-SF-iGluSnFR.A184V at DIV4 and imaged at DIV13 lookedqualitatively similar for both variants and as expected for a membranetargeted protein. To resolve fast stimulus associated changes influorescence, a time series of 100 frames at 60 Hz during a paired-pulsestimulation paradigm was acquired. Basal fluorescence before stimulationwas clearly stronger for A184V, the high affinity sensor, leading to ahigher SNR (FIG. 65A and FIG. 65C). However, when dividing each frame byan average of the pre-stimulus images for both variants of SF-iGluSnFRlocalized spots where fluorescence increases was observed (FIG. 65B,FIG. 65D, arrows), likely representing synaptic release sites. ROIs weredefined based on these spots, and fluorescence within these ROIs(background subtracted) was averaged for every image in the time series.

FIG. 66A-J Vesicle release sites can be localized by identifying thecenter of stimulus-evoked SF-iGluSnFR fluorescence changes. (FIGS. 66Aand 66B) Representative images of SF-iGluSnFR.S72A and SF-iGluSnFR.A184Vexpression in primary neuron cultures. Markers indicate the centers ofGaussians fitted to fluorescence profiles calculated across identifiedrelease sites from consecutive stimulation trials (such as shown in(FIG. 66E)). Note that the scatter of the centers of the localizedrelease sites is substantially larger for SF-iGluSnFR.A184V (16-25stimulation trials per experiment with inter-stimulus intervals of 20-60s, 20 frames before and 10 frames after stimulation were recorded).(FIGS. 66C and 66D) Spots of increased fluorescence as they occurimmediately after electrical stimulation when neurons are expressingSF-iGluSnFR.S72A or SF-iGluSnFR.A184V. 10 frames after the stimulus wereaveraged and divided by an average of 5 frames before stimulation. Inthis way, structures, which do not change fluorescence after simulation(background/inactive dendritic segments) will become 1. The lookup tableof these images was adjusted to range from 1 to 1.5. (FIG. 66E) Lineprofiles calculated across the response sites shown in (FIG. 66C) and(FIG. 66D) (dashed lines) and superimposed Gaussian fits (lines). Thewidth of the fitted Gaussian profiles were 0.57 and 1.11 μm forSF-iGluSnFR.S72A and SF-iGluSnFR.A184V, respectively. (FIG. 66F)Localization is more precise for SF-iGluSnFR. S72A. For each selectedresponding site (n=28-53), the mean deviation of the center of theGaussians across the stimulation trials was calculated. These valueswere averaged and bar graphed for each SF-iGluSnFR variant. (FIGS. 66Gand 66H) Width and amplitude of fitted Gaussian functions aresignificantly larger for the high affinity A184V sensor. (FIG. 661)Gaussian fits to profiles obtained from individual (not averaged) framesafter stimulus reveal the persistence of the SF-iGluSnFR.A184V variant.(FIG. 66J) Left: Gaussians fitted to the SF-iGluSnFR.A184V-mediatedsignal progressively broaden over time indicating that also sensormolecules remote to the site of release bind glutamate. Right: Same dataas on left, but plotted as width² over 4*t. The data points can beapproximated by a line consistent with a diffusional spread ofglutamate. The slope of the fitted line estimates the apparent(A184V-slowed) diffusion coefficient (D_(app)) of synaptically releasedglutamate to be 4.3 μm²/s in vitro. This value is orders of magnitudesmaller than the diffusion coefficient of free glutamate in solution(˜600-700 μm{circumflex over ( )}2/s) indicating that A184V not onlyprolongs but also substantially localizes glutamate molecules at thesites of release.

FIG. 67A-C Annotated amino acid sequences of SF-iGluSnFR (FIG. 67A),SF-Azurite-iGluSnFR (FIG. 67C), and SF-Venus-iGluSnFR (FIG. 67B).Domains colored as indicated. Affinity modulating mutations S72A andA184V/S are indicated by orange arrow. Mutations from SF-iGluSnFR toSF-Venus-iGluSnFR and SF-Azurite-iGluSnFR indicated in red.

FIG. 68A-D Characterization of soluble, purified SF-Azurite-iGluSnFR.(FIG. 68A) Titration of SF-Azurite-iGluSnFR yields a K_(d) of 62±11 μM,error bars are standard deviation of three measurements. Excitation(FIG. 67B), emission (FIG. 67C), and absorption (FIG. 68D) spectra ofSF-Azurite-iGluSnFR (light blue) and Azurite (dark blue), with glutamate(solid line) and without (dashed line).

FIG. 69A-D Spectra of SF-Venus-iGluSnFR. Excitation (FIG. 69A), emission(FIG. 69B), and absorbance (FIG. 69C) spectra of SF-Venus-iGluSnFR(yellow) with (solid line) and without (dashed line) glutamate. (FIG.69D) 2-photon spectrum with SF-iGluSnFR (black) and vertical 1030 nmmarkup included for reference.

FIG. 70A-F Annotated amino acid sequences of the SF biosensors disclosedherein. Affinity modulating mutations S72A and A184V/S are indicatedwith small case letters. For SEQ ID NOs: 176-182, each domain isindicated with underlining as follows:

; GltI 5-253; SF-GFP 147-238;

; SF-GFP 1-146; Glt1 254-279;

;

. SF-iGluSnFR.A184V (SEQ ID NO: 179); SF-iGluSnFR.A184S (SEQ ID NO:177); SF-iGluSnFR.S72A (SEQ ID NO:178); SF-Venus-iGluSnFR.A184V (SEQ IDNO: 179; mutations at residues T203Y and Y65G to shift the color and atresidues F46L and S72A to increase chromophore maturation are shown inlower case); SF-Venus-iGluSnFR.A184S (SEQ ID NO: 180; mutations atresidues T203Y and Y65G to shift the color and at residues F46L and S72Ato increase chromophore maturation are shown in lower case);SF-Venus-iGluSnFR.S72A (SEQ ID NO: 181; mutations at residues T203Y andY65G to shift the color and at residues F46L and S72A to increasechromophore maturation are shown in lower case); SF-Azurite-iGluSnFR(SEQ ID NO: 182; mutations at residues T65S and Y66H to shift the colorand at residues V150I and V224R to improve maturation and brightness areshown in lower case; Linker1 mutations: GltI-cpSFGFP connection fromPILVSHNV (SEQ ID NO: 187) to PILGYHNV (SEQ ID NO: 188); Linker2mutations: cpSFGFP-GltI connection from YNFNNPLN (SEQ ID NO: 189) toYNFNEQLN (SEQ ID NO: 190)); iDexSnFR (or SF-GlucoseSensor) (SEQ ID NO:183); iGABASnFR (SEQ ID NO: 184; cpSFGFP was inserted after D276 of thePf622 starting sequence. Insertion of cpSFGFP is after residue D276 ofPf622. Residues RS near the N-terminus encode BglII, and residues LQ atthe C-terminus encode PstI. Mutations included in iGABASnFR include:affinity modulating hinge mutation: Pf622: F101L; Pf622-SFGFP interface:Pf622: N260A; Linker 1: SHNVY (SEQ ID NO: 191) of SFGFP to LAQVR (SEQ IDNO: 192) (SFGFP: S147L, H148A, N149Q, Y151R); Linker 2: SFGFP (SEQ IDNO: 193): F145W; Linker 2: SVLAP (SEQ ID NO: 194) of Pf622 to ANLAP (SEQID NO: 195) (Pf622: S277A, V278N); Binding site mutation: Pf622:F102G/Y. Underlining indicates the domain as follows:

Pf622 2-276; SF-GFP 147-238; Linker; SF-GFP 1-146; Pf622 277-320;

Binding site mutation F102 indicated with a small case letter; andiAChSnFR (E. coli expression vector shown in SEQ ID NO:185 with thedomains indicated as follows:

Thermoanaerobacter sp. X513 choline binding protein sequence; Linkerregions; Circularly permuted super-folder EGFP;

and mammalian expression vector shown in SEQ ID NO: 186 with the domainsindicated as follows:

Thermoanaerobacter sp. X513 choline binding protein sequence; Linkerregions; Circularly permuted super-folder EGFP;

.

FIG. 71 A schematic of Structure I as described herein.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery ofstructures and methods related to and useful for genetically encodedbiosensors. Specifically, the disclosure provides genetically encodedrecombinant or chimeric peptides for use as biosensors and methods forthe design, production, and use of such biosensors. As described below,these sensors can be employed (e.g., expressed) in biological systems todetect and/or monitor a wide range of target analytes (e.g., a defined,selected, and/or specific analytes) due, in part, to the signal changegenerated by the sensors upon binding to their respective analyte(s),which signal change allows bound and unbound sensors to bedistinguished.

While the disclosure encompasses generic biosensors and methods relatedthereto, examples of particular binding sensors, including biosensorsfor detecting maltose, sucrose, maltotriose, glutamate, phosphonate, andglucose are also disclosed.

Compositions

Provided herein are genetically encoded biosensors, i.e., nucleic acidsencoding peptides, and/or the encoded peptides (e.g., isolatedpeptides), for use as biosensors. Biosensors herein include geneticallyencoded recombinant peptides containing an analyte-binding frameworkportion linked (e.g., operably linked) to at least one independentsignaling portion, wherein the independent signaling portion isallosterically modulated or regulated by the framework portion uponinteraction of the framework portion with an analyte (e.g., a defined,selected, and/or specific analyte), such that signaling from thesignaling portions is altered upon interaction of the framework portionwith the analyte.

In some instances, an independent signaling portion is present at a sitewithin the framework portion that undergoes a conformational change uponinteraction of the framework portion with an analyte such that theconformational change allosterically modulates or regulates signaling bythe signaling portion. For example, biosensors herein can includestructure I.

In some instances, signaling by the signaling portion is detectablyaltered upon interaction (e.g., binding) of the framework portion withan analyte. For example, signaling by the signaling portion candetectably increase or detectably decrease upon interaction (e.g.,binding) of the framework portion with an analyte. In some cases,biosensors have a signal change upon binding (e.g., specific binding) totheir respective analyte of at least about, for example, ±0.5, and/or anincrease or decrease in signal of at least about, for example, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, 750%, 1000%, ormore than 1000%, e.g., relative to unbound biosensor. In some increases,the level of signal change is linked to background signal. Valuesrepresented here can be converted and/or expressed into any conventionalunits using ordinary skill. For example, units can be expressed as‘signal change’ (as used above), ΔF/F and/or as signal-to-noise ratio(e.g., ΔF/F multiplied by the square root of the number of photonscollected). In some instances, signaling by a biosensor can be intensitybased.

In some instances, biosensors herein are distinguishable from Forsterresonance energy transfer, also known as fluorescence resonance energytransfer (FRET)-based sensors, which require donor and acceptorchromophores, e.g., that function in concert, in that they includeindependently functioning or detectable signaling portions. For example,in some instances, signaling by a first signaling portion of a biosensorherein is independent of signaling by a second signaling portion withinthe same or a distinct biosensor. As noted above, signaling portions areallosterically regulated by the framework portion to which they arelinked upon interaction of the framework portion with an analyte (e.g.,a defined, selected, and/or specific analyte).

Framework Portions

Framework portions include genetically encoded macromolecules (e.g.,proteins or peptides) that undergo conformational alteration (e.g., astructural change) upon interaction (e.g., binding) with, or to, ananalyte (e.g., an analyte-binding dependent conformational alteration).For example, genetically encoded framework portions can have a firststructure in the absence of an analyte (e.g., in an unbound or openstate) and a second structure, that is detectably distinct (e.g.,differences in structures before and after a conformational change canbe observed using methods known in the art) from the first structure, inthe presence of an analyte (e.g., in a bound or closed state), e.g.,under physiologic conditions. In some instances, the conformationalchange that occurs upon interaction with an analyte (e.g., ananalyte-binding dependent conformational alteration) is detectablydistinct (e.g., can be observed using methods known in the art) from aconformational change that may occur for the same protein or peptideunder other physiological conditions (e.g., a change in conformationinduced by altered temperature, pH, voltage, ion concentration,phosphorylation).

Methods for identifying proteins or peptides that exhibit suitableconformational characteristics and/or for observing differences instructure between structures or before and after a conformational changeare known in the art and/or are described herein. Such methods caninclude, for example, one or more of structural analysis,crystallography, NMR, EPR using Spin label techniques, CircularDichroism (CD), Hydrogen Exchange surface Plasmon resonance,calorimetry, and/or FRET.

In some instances, framework portions can have a first structure in theabsence of an analyte (e.g., in an unbound or open state) and a secondstructure, that is detectably distinct (e.g., can be observed usingmethods known in the art) from the first structure, in the presence ofan analyte (e.g., in a bound or closed state), e.g., under physiologicconditions, wherein the structural change between the open and closedstate can allosterically modulate an independent signaling portionrecombinantly (e.g., artificially introduced) present within theframework portion (see, e.g., Structure I).

Framework portions can also interact (e.g., bind) with at least oneanalyte (e.g., at least one defined, specific, and/or selected analyte).In some instances, a framework portion can interact specifically withone analyte (e.g., at least one defined, specific, and/or selectedanalyte). In such cases, affinity of binding between the frameworkbinding peptide and the analyte can be high or can be controlled (e.g.,with millimolar, micromolar, nanomolar, or picomolar affinity).Alternatively, the single framework binding protein can bind two or moreanalytes (e.g., two or more defined, specific, and/or selectedanalytes). In such cases, affinity of binding to the two or moreanalytes can be the same or distinct. For example, the affinity ofbinding can be greater for one analyte than it is for a second or third,etc., analyte. In some instances, binding between a framework portionand an analyte (e.g., at least one defined, specific, and/or selectedanalyte) have an affinity of for example, 10 mM to 1 pM.

As used herein, the term “analyte” can include naturally occurringand/or synthetic sugars, amino acids, proteins (e.g., proteins,peptides, and/or antibodies), hormones, ligands, chemicals (e.g., smallmolecules), pharmaceuticals, nucleic acids, cells, tissues, andcombinations thereof.

In some instances, biosensors can include one, two, or more frameworkbinding portions that bind (e.g., binds specifically) a single analyte(e.g., a single defined, specific, and/or selected analyte) or distinctanalytes (e.g., two or more distinct defined, specific and/or selectedanalytes). Alternatively or in addition, the framework portion can bechimeric. In such cases, a first part of the framework portion can be afirst peptide or can be derived from a first peptide, and a second partof the framework portion can be a second peptide or can be derived froma second peptide, wherein the first a second peptides are combined toresult in a single peptide.

Accordingly, framework portions can include macromolecules that undergoa conformational change upon interaction with an analyte. Onenon-limiting example of a suitable macromolecule is Calmodulin (CaM).CaM is in an extended shape in the absence of Ca²⁺ and in a condensedconformation in the presence of Ca²⁺ (Kuboniwa et al., Nat. Struc.Biol., 2:768-776, 1996 and Fallon and Quiocho, Structure, 11:1303-1307,2003).

In some instances, a framework binding portion can be a bacterialprotein or can be derived from a bacterial protein. Suitable bacterialproteins can include, but are not limited to, for example, periplasmicbinding proteins (PBPs).

PBPs from bacteria are generally useful in the biosensors herein atleast because they undergo dramatic conformational changes upon ligandbinding (Ouiocho et al. Mol. Microbiol., 20:17-225, 1996). X-ray crystalstructures of the apo (open) and bound (closed) forms of various PBPsreveal that these proteins have two (typically, although some have more)domains that undergo a large hinge-twist movement relative to each otherin a Venus flytrap manner (Dwyer and Hellinga, Curr. Opin. Struc. Biol.,14:495-504, 2004). This conformational change has been exploited tocreate a number of FRET-based genetically encoded sensors (see, e.g.,Deuschle et al., Pro. Sci, 14:2304-2314, 2005; Deuschle et al.,Cytometry, 64:3-9, 2005; Okumoto et al., Proc. Natl. Acad. Sci. USA.,102:8740-8745, 2005; Bogner and Ludewig, J. Fluoresc., 17:350-360, 2007;and Gu et al., FEBS Letters, 580:5885-5893, 2006). In addition, theligand-binding diversity of the PBP superfamily is large (Dwyer andHellinga, Curr. Opin. Struc. Biol., 14:495-504, 2004).

In some instances, framework portions can include, for example, one ormore of: arabinose-binding protein(s), glucose/galactose-bindingprotein(s), histidine-binding protein(s), maltose-binding protein(s),glutamine-binding protein(s), maltotriose-binding protein(s), RBP,ribose-binding protein(s), acetylcholine binding protein(s), cholinebinding protein(s), lysine binding protein(s), arginine bindingprotein(s), gamma aminobutyric acid (GABA) binding protein(s),ion-binding protein(s), peptide-binding protein(s), lactate-bindingprotein(s), histamine-binding protein(s), and/orLeucine/Isoleucine/Valine binding protein(s), including full lengthproteins, fragments, and/or variants thereof.

In some instances, exemplary framework portions can include: SEQ IDNO:105, which is Escherichia coli maltodextrin-binding protein (EcMBP)(UniProt accession number POAEX9); SEQ ID NO: 106, which is PyrococcusFuriosus maltotriose-binding protein (PfMBP) (UniProt accession numberP58300); SEQ ID NO:107, which is E. coli glutamate-binding protein(EcYbeJ) (UniProt accession number Q1R3F7); SEQ ID NO:108, which is E.coli phosphonate-binding protein (EcPhnD) (UniProt accession numberP37902); and/or SEQ ID NO:109, which is Thermus thermophilusglucose-binding protein (TtGBP) (UniProt accession number Q72KX2,including full length proteins, fragments, and/or variants thereof.

In some instances, exemplary framework portions can include SEQ ID NO:110 (UniProt accession number Q92N37); SEQ ID NO:111 (UniProt accessionnumber D0VWx8, SEQ ID NO:112 (UniProt accession number Q7CX36), and/orSEQ ID NO:113 (UniProt accession number POAD96, including full lengthproteins, fragments, and/or variants thereof.

In some embodiments, exemplary framework portions can include residues24-272 and 517-542 of SEQ ID NO: 176 (SF-iGluSnFR.A184V); residues24-272 and 517-542 of SEQ ID NO: 177 (SF-iGluSnFR.A184S); residues24-272 and 517-542 of SEQ ID NO: 178 (SF-iGluSnFR.S72A); residues 24-272and 517-542 of SEQ ID NO: 179 (SF-Venus-iGluSnFR.A184V); residues 24-272and 517-542 of SEQ ID NO: 180 (SF-Venus-iGluSnFR.A184S); residues 24-272and 517-542 of SEQ ID NO: 181 (SF-Venus-iGluSnFR.S72A); residues 24-271and 519-541 of SEQ ID NO: 182 (SF-Azurite-iGluSnFR); residues 24-350 and595-664 of SEQ ID NO: 183 (iDexSnFR or SF-GlucoseSensor); residues24-298 and 543-586 of SEQ ID NO: 184 (iGABASnFR); residues 25-99 and348-545 of SEQ ID NO: 185 (iAChSnFR E. coli expression sequence); orresidues 24-98 and 347-544 of SEQ ID NO: 186 (iAChSnFR mammalianexpression sequence).

In some instances, framework portions, or biosensors, do not includesignal peptides, or portions of signal peptides, that would otherwise bepresent in the peptide from which the framework portion is derived.

Signaling Portions

Biosensors herein include one or more genetically encoded signalingportions (e.g., independent signaling portions) within the amino acidsequence of a framework portion at a site(s) within the frameworkportion that undergo(es) a conformational change upon interaction of theframework portion with an analyte (e.g., a defined, specific, and/orselected analyte).

Signaling portions (e.g., independent signaling portions) includegenetically encoded molecules (e.g., peptides or proteins) that can beallosterically induced to emit a detectable signal (e.g., ananalyte-binding dependent signal).

In some instances, the detectable signal is detectably distinct (e.g.,can be distinguished using methods known in the art and/or disclosedherein) from a signal emitted by the molecule prior to allostericinducement (e.g., signaling portions can emit a detectable signal in twodetectably distinct states. For example, first signal can be emitted inunbound state and a second signal can be emitted in bound state). Asnoted above, in some instances, the detectable signal is proportional tothe degree of allosteric inducement. In some instances, if two or moresignaling portions are present in a biosensor, then two or moredetectably distinct signals can be emitted by the biosensor.

In some instances, a genetically encoded independent signaling portionis a genetically encoded fluorescent protein (FP), e.g., a macromoleculecontaining a functional group (e.g., a fluorophore) that absorbs energyof a specific wavelength and re-emits energy at a different (but equallyspecific) wavelength, including, for example, circularly permuted FP(cpFP). In some instances, a signaling portion is a “superfolder” FP(e.g., Pedelacq et al., 2006, Nat. Biotech., 24:79-88), e.g., acircularly permuted SF FP.

As used herein, the term “fluorophore” relates to a functional group ina molecule which will absorb energy of a specific wavelength and re-emitenergy at a different (but equally specific) wavelength. In someinstances, fluorophore containing molecules include fluorescentproteins. The fluorophore in green fluorescent protein (GFP) includesSer-Tyr-Gly sequence (i.e., Ser65-dehydroTyr66-Gly67), which ispost-translationally modified to a4-(p-hydroxybenzylidene)-imidazolidin-5. Exemplary genetically encodedfluorescent proteins include, but are not limited to, fluorescentproteins from coelenterate marine organisms, e.g., Aequorea victoria,Trachyphyllia geoffroyi, coral of the Discosoma genus, Rennilla mulleri,Anemonia sulcata, Heteractis crispa, Entacmaea quadricolor, and/or GFP(including the variants S65T and EGFP, Rennilla mulleri GFP), cyanfluorescent protein (CFP), including Cerulean, and mCerulean3 (describedby Markwardt et al., PLoS ONE, 6(3)e17896.doi:10.1371/journal.pone.0017896), CGFP (CFP with Thr203Tyr: Hasan excitation and emission wavelength that is intermediate between CFPand EGFP), yellow fluorescent protein (YFP, e.g.,GFP-Ser65Gly/Ser72Ala/Thr203Tyr; YFP (e.g.,GFP-Ser65Gly/Ser72Ala/Thr203Tyr) with Va168Leu/Gln69Lys); Citrine (i.e.,YFP-Va168Leu/Gln69Met), Venus (i.e.,YFP-Phe46Leu/Phe64Leu/Met153Thr/Val163Ala/Ser175Gly), PA-GFP (i.e.,GFP-Val/163Ala/Thr203His), Kaede), red fluorescent protein (RFP, e.g.,long wavelength fluorescent protein, e.g., DsRed (DsRed1, DsRed2,DsRed-Express, mRFP1, drFP583, dsFP593, asFP595), eqFP611, and/or otherfluorescent proteins known in the art (see, e.g., Zhang et al., NatureReviews, Molecular and Cellular Biology, 3:906-908, 2002).

As set forth above, in some instances, fluorophore containing moleculesinclude fluorescent proteins that can be or that are circularlypermutated. Circular permutation methods are known in the art (see,e.g., Baird et al., Proc. Natl. Acad. Sci., 96:11241-11246, 1999; Topelland Glockshuber, Methods in Molecular Biology, 183:31-48, 2002) as are“superfolder” (SF) proteins (e.g., Pedelacq et al., 2006, Nat. Biotech.,24:79-88) (e.g., circularly permuted SF proteins).

In some instances, single-FP sensors have a number of advantages: theypreserve spectral bandwidth for multi-analyte imaging; their saturatedstates may be nearly as bright as the parental FP, and their ligand-freestates may be arbitrarily dim, providing large theoretical fluorescenceincreases. This allows for much greater changes in fluorescence and thusincreased signal-to-noise ratios and greater resistance tophotobleaching artifacts (Tian et al., Nat. Methods, 6:875-881, 2009).

In some instances, issues arising from long-term effects such as generegulation and protein expression and degradation can be identified bysimply fusing the intensity-based sensor to a another fluorescentprotein of different color, to serve as a reference channel.

In some instances, biosensors can include circularly permuted YFP(cpYFP) as a cpFP. cpYFP has been used as a reporter element in thecreation of sensors for H₂O₂ (HyPer) (Belousov et al., Nat. Methods,3:281-286, 2006), cGMP (FlincG) (Nausch et al., Proc. Natl. Acad. Sci.USA., 105:365-370, 2008), ATP:ADP ratio (Perceval) (Berg et al., Nat.Methods., 105:365-370, 2008), and calcium ions (Nakai et al., Nat.Biotechno., 19:137-141, 2001), including full length, fragments, and/orvariants thereof.

In some embodiments, exemplary sensor portions can include residues273-516 of SEQ ID NO: 176 (SF-iGluSnFR.A184V); residues 273-516 of SEQID NO: 177 (SF-iGluSnFR.A184S); residues 273-516 of SEQ ID NO: 178(SF-iGluSnFR.S72A); residues 273-516 of SEQ ID NO: 179(SF-Venus-iGluSnFR.A184V); residues 273-516 of SEQ ID NO: 180(SF-Venus-iGluSnFR.A184S); residues 273-516 of SEQ ID NO: 181(SF-Venus-iGluSnFR.S72A); residues 272-518 of SEQ ID NO: 182(SF-Azurite-iGluSnFR); residues 351-594 of SEQ ID NO: 183 (iDexSnFR orSF-GlucoseSensor); residues 299-544 of SEQ ID NO: 184 (iGABASnFR);residues 104-343 of SEQ ID NO: 185 (iAChSnFR E. coli expressionsequence); or residues 103-342 of SEQ ID NO: 186 (iAChSnFR mammalianexpression sequence).

Linker Portions

As shown in Structure I, biosensors herein can optionally include one ormore genetically encoded linkers positioned between or operably linkingthe framework portion and the signaling portion. Linker portions caninclude at least one naturally occurring or synthetic amino acid(discussed below) as exemplified by SEQ ID NOs: 9-49, 54-61, 64-76,79-90, 95-104. In some instances, linker can include one or more of SEQID NOs: 9-49, 54-61, 64-76, 79-90, 95-104, and/or portions of SEQ IDNOs: 9-49, 54-61, 64-76, 79-90, 95-104. For example, linkers caninclude, but are not limited to, one or more of: PxSHNVY (SEQ IDNO:114), xPSHNVY (SEQ ID NO:115), xxSHNVY (SEQ ID NO:116), xxSHNVF (SEQID NO:117), PxSHNVF (SEQ ID NO:118), PxSYNVF (SEQ ID NO:119), xxSYNVF(SEQ ID NO:120), PxSYNVF (SEQ ID NO:121), xxSYNVF (SEQ ID NO:122),PxSxNVY (SEQ ID NO:123), PxSHxVY (SEQ ID NO:124), PxSHNxY (SEQ IDNO:125), PxSHNVx (SEQ ID NO:126), FNxxY (SEQ ID NO:127), FNxY (SEQ IDNO:128), FNY (SEQ ID NO:129), FxY (SEQ ID NO:130), xxY (SEQ ID NO:131),WxY (SEQ ID NO:132), xKY, (SEQ ID NO:133), FNPxY (SEQ ID NO:134), FNxPY(SEQ ID NO:135), HNS (SEQ ID NO:136), GGS (SEQ ID NO:137), xxS (SEQ IDNO:138), xxK (SEQ ID NO:139), GGK (SEQ ID NO:140), PXS (SEQ ID NO:141),xPS (SEQ ID NO:142), Px (SEQ ID NO:143), xP (SEQ ID NO:144), IxxS (SEQID NO:145), NxPK (SEQ ID NO:146), NPcK (SEQ ID NO:147), PPxSH (SEQ IDNO:148), PPxxSH (SEQ ID NO:149), PPPxSH (SEQ ID NO:150), PPxPSH (SEQ IDNO:151), xxSH (SEQ ID NO:152), PPxx (SEQ ID NO:153), FNxKN (SEQ IDNO:154), FNxxKN (SEQ ID NO:155), FNxPKN (SEQ ID NO:156), FNPxKN (SEQ IDNO:157), FNxx (SEQ ID NO:158), N, ADGSSH (SEQ ID NO:159), ADxxSH (SEQ IDNO:160), ADxPSH (SEQ ID NO:161), ADPxSH (SEQ ID NO:162), ADxx (SEQ IDNO:163), ADxxSH (SEQ ID NO:164), FNPG (SEQ ID NO:165), FNxxPG (SEQ IDNO:166), xxPG (SEQ ID NO:167), FNxx (SEQ ID NO:168), FNPx (SEQ IDNO:169), KYxxSH (SEQ ID NO:170), KYPxSH (SEQ ID NO:171), KYxPSH (SEQ IDNO:172), FxxP (SEQ ID NO:173), FNxP (SEQ ID NO:174), and/or FNPx (SEQ IDNO:175), where “x” indicates any amino acid.

In some embodiments, exemplary linker portions can include residues365-370 of SEQ ID NO: 176 (SF-iGluSnFR.A184V); residues 365-370 of SEQID NO: 177 (SF-iGluSnFR.A184S); residues 365-370 of SEQ ID NO: 178(SF-iGluSnFR.S72A); residues 365-370 of SEQ ID NO: 179(SF-Venus-iGluSnFR.A184V); residues 365-370 of SEQ ID NO: 180(SF-Venus-iGluSnFR.A184S); residues 365-370 of SEQ ID NO: 181(SF-Venus-iGluSnFR.S72A); residues 365-370 of SEQ ID NO: 182(SF-Azurite-iGluSnFR); residues 443-448 of SEQ ID NO: 183 (iDexSnFR orSF-GlucoseSensor); residues 391-396 of SEQ ID NO: 184 (iGABASnFR);residues 100-103 and 344-347 of SEQ ID NO: 185 (iAChSnFR E. coliexpression sequence); or residues 99-102 and 343-346 of SEQ ID NO: 186(iAChSnFR mammalian expression sequence).

Exemplary Biosensor Constructs

As noted above, biosensors herein include genetically encodedbiosensors, i.e., nucleic acids encoding biosensors, and/or the encodedbiosensors (e.g., isolated biosensors), for use as biosensors. In someinstances, nucleic acids encoding biosensors include isolated nucleicacids. In some instances, the portion of a nucleic acid encoding abiosensor can include a single reading frame encoding the biosensor. Forexample, a biosensor can be encoded by a portion of a nucleic acid thatfalls within a start codon and a stop codon. In some instances,biosensors are isolated (e.g., biosensors are substantially free ofcontaminating and/or non-biosensor components).

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of:arabinose-binding protein(s), glucose/galactose-binding protein(s),histidine-binding protein(s), maltose-binding protein(s),maltotriose-binding protein(s), glutamine-binding protein(s), RBP,ribose-binding protein(s), acetylcholine binding protein(s), cholinebinding protein(s), lysine binding protein(s), arginine bindingprotein(s), gamma aminobutyric acid (GABA) binding protein(s),ion-binding protein(s), peptide-binding protein(s), lactate-bindingprotein(s), histamine-binding protein(s), and/orLeucine/Isoleucine/Valine binding protein(s), including full lengthproteins, fragments, and/or variants thereof, including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of: SEQ ID NO:105,which is Escherichia coli maltodextrin-binding protein (EcMBP) (UniProtaccession number POAEX9); SEQ ID NO: 106, which is Pyrococcus Furiosusmaltose-binding protein (PfMBP) (UniProt accession number P58300); SEQID NO:107, which is E. coli glutamate-binding protein (EcYbeJ) (UniProtaccession number Q1R3F7); SEQ ID NO:108, which is E. coliphosphonate-binding protein (EcPhnD) (UniProt accession number P37902);and/or SEQ ID NO:109, which is Thermus thermophilus glucose-bindingprotein (TtGBP) (UniProt accession number Q72KX2), including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of: SEQ ID NO: 110(UniProt accession number Q92N37); SEQ ID NO:111 (UniProt accessionnumber DOVWx8, SEQ ID NO:112 (UniProt accession number Q7CX36), and/orSEQ ID NO:113 (UniProt accession number POAD96), including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors include any one or more:

Maltose biosensors SEQ ID NOs: 1-8 (e.g., Escherichia colimaltodextrin-binding protein (EcMBP)) or SEQ ID NOs: 50-53 (e.g.,Pyrococcus furiosus maltose-binding protein (PfMBP)), including fulllength proteins, fragments and/or variants thereof;

Glutamate biosensors SEQ ID NOs: 62-63 (e.g., E. coli glutamate-bindingprotein (EcYbeJ)) or SEQ ID NOs: 176-182, including full lengthproteins, fragments and/or variants thereof;

Phosphonate biosensors SEQ ID NOs: 77-78 (e.g., E. coliphosphonate-binding protein (EcPhnD)), including full length proteins,fragments and/or variants thereof;

Glucose biosensors SEQ ID NOs: 91-94 (e.g., Thermus thermophilusglucose-binding protein (TtGBP)) and SEQ ID NO: 183, including fulllength proteins, fragments and/or variants thereof;

GABA biosensors SEQ ID NO: 184, including full length proteins,fragments and/or variants thereof; and/or

ACh biosensors SEQ ID NOs: 185 & 186, including full length proteins,fragments and/or variants thereof.

In some instances, nucleic acids encoding, and/or amino acid sequencesof, any of the framework portions, signaling portions, linker portions,or the entire biosensor sequence (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186)(e.g., any amino acid sequence) disclosed herein can be modified togenerate fragments (e.g., truncated peptides) and/or variants (e.g.,peptides with a defined sequence homology to the peptides disclosedherein). Variants can include framework portions, signaling portions,linker portions, or biosensors with amino acid sequences with homologyto the framework portions, signaling portions, linker portions, orbiosensors disclosed herein and/or truncated forms of the frameworkportions, signaling portions, linker portions, or biosensors herein. Insome instances, truncated forms of the framework portions, signalingportions, linker portions, or biosensors herein can include 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 50-100, 101-150, fewer amino acidsthan the framework portions, signaling portions, linker portions, and/orbiosensors herein, e.g., wherein the truncated biosensor variants retainat least at portion of the binding and/or signaling properties of samebiosensor without truncation (e.g., at least 50%, 60%, 70%, 80%, 90%, or100% of the binding and/or signaling properties of the same biosensorwithout truncation). In addition, truncations can be made at theamino-terminus, the carboxy-terminus, and/or within the body of theframework portions, signaling portions, linker portions, and/orbiosensors herein.

While variants are generally observed and discussed at the amino acidlevel, the actual modifications are typically introduced or performed atthe nucleic acid level. For example, variants with 95%, 96%, 97%, 98, or99% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186 can be generated bymodifying the nucleic acids encoding SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186 usingtechniques (e.g., cloning techniques) known in the art and/or that aredisclosed herein.

As with all peptides, polypeptides, and proteins, including fragmentsthereof, it is understood that modifications to the amino acid sequencecan occur that do not alter the nature or function of the peptides,polypeptides, or proteins. Such modifications include conservative aminoacids substitutions and are discussed in greater detail below.

The peptides, polypeptides, and proteins, including fragments thereof,provided herein are biosensors whose activity can be tested or verified,for example, using the in vitro and/or in vivo assays described herein.

In some instances, any of the framework portions, signaling portions, orthe biosensor sequence (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50,51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186) (e.g., anyamino acid sequence) described herein can be modified and varied so longas their desired function is maintained. For example, the polypeptidescan be modified as long as the resulting variant polypeptides have thesame or better characteristics as the polypeptide from which theyderived. For example, the variants can have the same or better affinityfor their respective analyte.

In some instances, the interacting face of a modified peptide can be thesame (e.g., substantially the same) as an unmodified peptide (methodsfor identifying the interacting face of a peptide are known in the art(Gong et al., BMC: Bioinformatics, 6:1471-2105 (2007); Andrade and Weiet al., Pure and Appl. Chem., 64(11):1777-1781 (1992); Choi et al.,Proteins: Structure, Function, and Bioinformatics, 77(1):14-25 (2009);Park et al., BMC: and Bioinformatics, 10:1471-2105 (2009)), e.g., tomaintain binding to an analyte. Alternatively, amino acids within theinteracting face can be modified, e.g., to decrease binding to ananalyte and/or to change analyte specificity.

The interacting face of a peptide is the region of the peptide thatinteracts or associates with other molecules (e.g., other proteins).Generally, amino acids within the interacting face are naturally morehighly conserved than those amino acids located outside the interactingface or interface regions of a protein. In some instances, an amino acidwithin the interacting face region of any of the framework portions orthe biosensor sequence (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50,51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186) (e.g., anyamino acid sequence) disclosed herein can be the same as the amino acidshown in any of the framework portions or the biosensor sequence (e.g.,SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91,92, 93, 94, and/or 176-186) (e.g., any amino acid sequence) disclosedherein or can be include conservative amino acid substitutions. In someinstances, an amino acid within the interacting face region any of theframework portions or the biosensor sequence (e.g., SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or176-186) (e.g., any amino acid sequence) disclosed herein can besubstituted with an amino acid that increases the interaction betweenthe framework portion or the biosensor sequence (e.g., SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or176-186) (e.g., any amino acid sequence) and an analyte.

In some instances, genetically encoded biosensors can include peptidesthat have at least 80, 85, 90, 95, 96, 97, 98, 99 percent identity tothe framework portions, signaling portions, or the biosensor sequence(e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, 94, and/or 176-186) (e.g., any amino acid sequence)described herein. Those of skill in the art readily understand how todetermine the identity of two polypeptides. For example, the identitycan be calculated after aligning the two sequences so that the identityis at its highest level.

Another way of calculating identity can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Adv.Appl. Math, 2:482 (1981), by the identity alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of identity can be obtained for nucleic acids by, forexample, the algorithms disclosed in Zuker, Science 244:48-52 (1989);Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-10 (1989); Jaeger etal., Methods Enzymol. 183:281-306 (1989), which are herein incorporatedby reference for at least material related to nucleic acid alignment. Itis understood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity and to be disclosed herein.

Amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional, or deletional modifications.Insertions include amino and/or terminal fusions as well asintra-sequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. Amino acid substitutions are typically of singleresidues, but can occur at a number of different locations at once;insertions usually will be on the order of about from 1 to 10 amino acidresidues; and deletions will range about from 1 to 30 residues.Deletions or insertions can be made in adjacent pairs, i.e., a deletionof 2 residues or insertion of 2 residues. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal construct. The mutations must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. Substitutional modifications arethose in which at least one residue has been removed and a differentresidue inserted in its place. In some instances, substitutions can beconservative amino acid substitutions. In some instances, variantsherein can include one or more conservative amino acid substitutions.For example, variants can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, or 40-50 conservativeamino acid substitutions. Alternatively, variants can include 50 orfewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 9 or fewer, 8or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or2 or fewer conservative amino acid substitutions. Such substitutionsgenerally are made in accordance with the following Table 1 and arereferred to as conservative substitutions. Methods for predictingtolerance to protein modification are known in the art (see, e.g., Guoet al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004)).

TABLE 1 Conservative Amino Acid Substitutions Amino Acid Substitutions(others are known in the art) Ala Ser, Gly, Cys Arg Lys, Gln, His AsnGln, His, Glu, Asp . . . Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn,Lys, Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro, Ala, Ser His Asn, Gln, LysIle Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, His Met Leu,Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His Ser Thr, Cys, Ala ThrSer, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

In some instances, substitutions are not conservative. For example, anamino acid can be replaced with an amino acid that can alter someproperty or aspect of the peptide. In some instances, non-conservativeamino acid substitutions can be made, e.g., to change the structure of apeptide, to change the binding properties of a peptide (e.g., toincrease or decrease the affinity of binding of the peptide to ananalyte and/or to alter increase or decrease the binding specificity ofthe peptide).

Modifications, including the specific amino acid substitutions, are madeby known methods. By way of example, modifications are made bysite-specific mutagenesis of nucleotides in the DNA encoding theprotein, thereby producing DNA encoding the modification, and thereafterexpressing the DNA in recombinant cell culture. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example M13 primer mutagenesis and PCRmutagenesis.

Nucleic Acids

The disclosure also features nucleic acids encoding the biosensors(e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, 94, and/or 176-186) described herein, including variantsand/or fragments of the biosensors (e.g., variants and/or fragments ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91,92, 93, 94, and/or 176-186). These sequences include all degeneratesequences related to the specific polypeptide sequence, i.e., allnucleic acids having a sequence that encodes one particular polypeptidesequence as well as all nucleic acids, including degenerate nucleicacids, encoding the disclosed variants and derivatives of thepolypeptide sequences. Thus, while each particular nucleic acid sequencemay not be written out herein, it is understood that each and everysequence is in fact disclosed and described herein through the disclosedpolypeptide sequences.

In some instances, nucleic acids can encode biosensors with 95, 96, 97,98, or 99 identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186.

In some instances, nucleic acids can encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 20-30, 30-40, or 40-50 conservative amino acidsubstitutions.

In some instances, nucleic acids can encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186containing 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 orfewer, 3 or fewer, or 2 or fewer conservative amino acid substitutions

Also provided herein are vectors comprising the biosensors (e.g, SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,94, and/or 176-186) described herein, including variants and/orfragments of the biosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186). Forexample:

Vectors can include nucleic acids that encode biosensors with 95, 96,97, 98, or 99 identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186.

Vectors can include nucleic acids that encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 20-30, 30-40, or 40-50 conservative amino acidsubstitutions.

Vectors can include nucleic acids that encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186containing 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 orfewer, 3 or fewer, or 2 or fewer conservative amino acid substitutions

Examples of suitable vectors include, but are not limited to, plasmids,artificial chromosomes, such as BACs, YACs, or PACs, and viral vectors.As used herein, vectors are agents that transport the disclosed nucleicacids into a cell without degradation and, optionally, include apromoter yielding expression of the nucleic acid molecule in the cellsinto which it is delivered.

Viral vectors can include, for example, Adenovirus, Adeno-associatedvirus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNAviruses, including these viruses with the HIV backbone. Any viralfamilies which share the properties of these viruses which make themsuitable for use as vectors are suitable. Retroviral vectors, in generalare described by Coffin et al., Retroviruses, Cold Spring HarborLaboratory Press (1997), which is incorporated by reference herein forthe vectors and methods of making them. The construction ofreplication-defective adenoviruses has been described (Berkner et al.,J. Virology 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83(1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al.,J. Virology 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72(1993)). Recombinant adenoviruses have been shown to achieve highefficiency after direct, in vivo delivery to airway epithelium,hepatocytes, vascular endothelium, CNS parenchyma, and a number of othertissue sites. Other useful systems include, for example, replicating andhost-restricted non-replicating Vaccinia virus vectors.

Non-viral based vectors can include expression vectors comprisingnucleic acid molecules and nucleic acid sequences encoding polypeptides,wherein the nucleic acids are operably linked to an expression controlsequence. Suitable vector backbones include, for example, thoseroutinely used in the art such as plasmids, artificial chromosomes,BACs, YACs, or PACs. Numerous vectors and expression systems arecommercially available from such corporations as Novagen (Madison,Wis.), Clontech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), andInvitrogen/Life Technologies (Carlsbad, Calif.). Vectors typicallycontain one or more regulatory regions. Regulatory regions include,without limitation, promoter sequences, enhancer sequences, responseelements, protein recognition sites, inducible elements, protein bindingsequences, 5′ and 3′ untranslated regions (UTRs), transcriptional startsites, termination sequences, polyadenylation sequences, and introns.

Promoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as polyoma,

Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, andmost preferably cytomegalovirus (CMV), or from heterologous mammalianpromoters, e.g. β-actin promoter or EF1α promoter, or from hybrid orchimeric promoters (e.g., CMV promoter fused to the β-actin promoter).Of course, promoters from the host cell or related species are alsouseful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′ or3′ to the transcription unit. Furthermore, enhancers can be within anintron as well as within the coding sequence itself. They are usuallybetween 10 and 300 base pairs in length, and they function in cis.Enhancers usually function to increase transcription from nearbypromoters. Enhancers can also contain response elements that mediate theregulation of transcription. While many enhancer sequences are knownfrom mammalian genes (globin, elastase, albumin, fetoprotein, andinsulin), enhancers derived from a eukaryotic cell viruses can be used.Examples of such can include the SV40 enhancer on the late side of thereplication origin, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promoter and/or the enhancer can be inducible (e.g. chemically orphysically regulated). A chemically regulated promoter and/or enhancercan, for example, be regulated by the presence of alcohol, tetracycline,a steroid, or a metal. A physically regulated promoter and/or enhancercan, for example, be regulated by environmental factors, such astemperature and light. Optionally, the promoter and/or enhancer regioncan act as a constitutive promoter and/or enhancer to maximize theexpression of the region of the transcription unit to be transcribed. Incertain vectors, the promoter and/or enhancer region can be active in acell type specific manner. Optionally, in certain vectors, the promoterand/or enhancer region can be active in all eukaryotic cells,independent of cell type. Promoters of this type can include the CMVpromoter, the SV40 promoter, the β-actin promoter, the EF1α promoter,and the retroviral long terminal repeat (LTR).

The provided vectors also can include, for example, origins ofreplication and/or markers. A marker gene can confer a selectablephenotype, e.g., antibiotic resistance, on a cell. The marker product isused to determine if the vector has been delivered to the cell and oncedelivered is being expressed. Examples of selectable markers formammalian cells are dihydrofolate reductase (DHFR), thymidine kinase,neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin.When such selectable markers are successfully transferred into amammalian host cell, the transformed mammalian host cell can survive ifplaced under selective pressure. Examples of other markers include, forexample, the E. coli lacZ gene, green fluorescent protein (GFP), andluciferase. In addition, an expression vector can include a tag sequencedesigned to facilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such as GFP,glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide including at either the carboxyl oramino terminus.

The disclosure further provides cells comprising the biosensors (e.g.,SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91,92, 93, 94, and/or 176-186) described herein, including variants and/orfragments of the biosensors (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186). Cellscan include, for example, eukaryotic and/or prokaryotic cells. Forexample, cells can include, but are not limited to cells of E. coli,Pseudomonas, Bacillus, Streptomyces; fungi cells such as yeasts(Saccharomyces, and methylotrophic yeast such as Pichia, Candida,Hansenula, and Torulopsis); and animal cells, such as CHO, R1.1, B-W andLM cells, African Green Monkey kidney cells (for example, COS 1, COS 7,BSC1, BSC40, and BMT10), insect cells (for example, Sf9), human cellsand plant cells. Suitable human cells can include, for example, HeLacells or human embryonic kidney (HEK) cells. In general, cells that canbe used herein are commercially available from, for example, theAmerican Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va.20108. See also F. Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., (1998).

Optionally, the biosensors (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186)described herein, including variants and/or fragments of the biosensors(e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, 94, and/or 176-186) can be located in the genome of thecell (e.g., can be stably expressed in the cell) or can be transientlyexpressed in the cell.

Methods of making the provided cells are known and the method oftransformation and choice of expression vector will depend on the hostsystem selected. Transformation and transfection methods are described,e.g., in F. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., (1998), and, as described above,expression vectors may be chosen from examples known in the art.

There are a number of compositions and methods which can be used todeliver the nucleic acid molecules and/or polypeptides to cells, eitherin vitro or in vivo via, for example, expression vectors. These methodsand compositions can largely be broken down into two classes: viralbased delivery systems and non-viral based deliver systems. Such methodsare well known in the art and readily adaptable for use with thecompositions and methods described herein.

By way of example, the provided polypeptides and/or nucleic acidmolecules can be delivered via virus like particles. Virus likeparticles (VLPs) consist of viral protein(s) derived from the structuralproteins of a virus. Methods for making and using virus like particlesare described in, for example, Garcea and Gissmann, Current Opinion inBiotechnology 15:513-7 (2004). The provided polypeptides can bedelivered by subviral dense bodies (DBs). DBs transport proteins intotarget cells by membrane fusion. Methods for making and using DBs aredescribed in, for example, Pepperl-Klindworth et al., Gene Therapy10:278-84 (2003). The provided polypeptides can be delivered by tegumentaggregates. Methods for making and using tegument aggregates aredescribed in International Publication No. WO 2006/110728.

Also provided are transgenic animals comprising one or more cells thebiosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62,63, 77, 78, 91, 92, 93, 94, and/or 176-186) described herein, includingvariants and/or fragments of the biosensors (e.g, SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or176-186). As used herein, the term animal refers to non-human animals,including, mammals, amphibians and birds. Specifically, examples includesheep, feline, bovines, ovines, pigs, horses, rabbits, guinea pigs,mice, hamsters, rats, non-human primates, and the like. As used herein,transgenic animal refers to any animal, in which one or more of thecells of the animal contain a heterologous nucleic acid. Theheterologous nucleic acid can be introduced using known transgenictechniques. The nucleic acid is introduced into the cell, directly orindirectly. For example, the nucleic acid can be introduced into aprecursor of the cell or by way of deliberate genetic manipulation, suchas by microinjection or by infection with a recombinant virus. Thenucleic acid may be integrated within a chromosome, or it may be anextrachromosomally replicating DNA.

Methods for making transgenic animals using a variety of transgenes havebeen described in Wagner et al. (1981) Proc. Nat. Acad. Sci. USA,78:5016-5020; Stewart et al. (1982) Science, 217:1046-1048; Constantiniet al. (1981) Nature, 294:92-94; Lacy et al. (1983) Cell, 34:343-358;McKnight et al. (1983) Cell, 34:335-341; Brinstar et al. (1983) Nature,306:332-336; Palmiter et al. (1982) Nature, 300:611-615; Palmiter et al.(1982) Cell, 29:701-710; and Palmiter et al. (1983) Science,222:809-814. Such methods are also described in U.S. Pat. Nos.6,175,057; 6,180,849; and 6,133,502.

By way of example, the transgenic animal can be created by introducing anucleic acid into, for example, an embryonic stem cell, an unfertilizedegg, a fertilized egg, a spermatozoon or a germinal cell containing aprimordial germinal cell thereof, preferably in the embryogenic stage inthe development of a non-human mammal (more preferably in thesingle-cell or fertilized cell stage and generally before the 8-cellphase). The nucleic acid can be introduced by known means, including,for example, the calcium phosphate method, the electric pulse method,the lipofection method, the agglutination method, the microinjectionmethod, the particle gun method, the DEAE-dextran method and other suchmethod. Optionally, the nucleic acid is introduced into a somatic cell,a living organ, a tissue cell or other cell by gene transformationmethods. Cells including the nucleic acid may be fused with theabove-described germinal cell by a commonly known cell fusion method tocreate a transgenic animal.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g., mouse, rat,guinea pig, and the like. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. Aftertransformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the nucleic acid. Those colonies thatare positive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting littersscreened for mutant cells having the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected. The chimeric animals are screened for thepresence of the nucleic acid, and males and females having themodification are mated to produce homozygous progeny transgenic animals.

Kits comprising one or more containers and the nucleic acid sequences,polypeptides, vectors, cells, provided herein, or combinations thereof,are also provided. For example, provided is a kit comprising (i) anucleic acid sequence encoding a biosensor described herein (e.g, one ormore of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, 94, and/or 176-186), including variants and/or fragmentsof the biosensor (e.g, variants or fragments of one or more of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,94, and/or 176-186), (ii) a polypeptide comprising a biosensor describedherein (e.g, one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186), includingvariants and/or fragments of the biosensor (e.g, variants or fragmentsof one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53,62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186), (iii) a vectorcomprising the nucleic acid of (i), (iv) a cell comprising the nucleicacid or (i) and/or the polypeptide of (ii), (v) a cell comprising thevector of (iii). The kit can comprise any combination of (i)-(v).Optionally, the kit further comprises reagents for using the nucleicacid or peptide biosensors, vectors, and/or cells. For example, if thekit comprises cells, the kit may also comprise cell culture medium.Optionally, the kit further comprises instructions for use. Optionally,the kit further comprises a GPCR, a GPCR-encoding nucleic acid sequence.

Design and Production/Manufacture Methods

Using the methods described herein, it is possible to design, produce,and/or adapt genetically encoded biosensors to assays for a variety ofclasses of analytes. The provided materials and methods facilitate thediscovery of new compounds targeting a wide array of protein targets,including but not limited to: endogenous targets responsible for diseasestate progression, targets on pathogens for treating infectiousdiseases, and endogenous targets to be avoided (thus screening early forpotential drug side effects and toxicity).

Methods herein provide systematic and generic approaches for the designand production of genetically encoded recombinant peptides containing ananalyte-binding framework portion linked (e.g., operably linked) to asignaling portion, wherein the signaling portion is allostericallymodulated or regulated by the framework portion upon interaction of theframework portion with an analyte. Generally, methods include: (i)selecting one or more target analytes; (ii) selecting a frameworkportion (e.g., a PBP) that interacts with (e.g., interacts specificallywith) or binds to (e.g., binds specifically to) the target analyte andthat undergoes a conformational change upon interacting with or bindingto the analyte; (iii) identifying sites or amino acid positions withinthe framework portion (e.g., the PBP) where the conformational changeoccurs; and (iv) inserting or cloning a signaling portion into the siteor amino acid position identified in (iii). Methods can, optionally,further include: (v) modifying or optimizing linker sequences betweenthe framework portion and the signaling portion, for example, by geneticmanipulation (e.g., by point mutation); (vi) modifying or optimizinganalyte binding; (vii) modifying the signal generated by the biosensor;and/or (viii) cloning the biosensor into a suitable vector.

In some instances: (iii) includes identification of insertion sites byanalysis of the structure (e.g., crystal structure) of the selectedframework portion (e.g., the selected PBP) in one or both of its openand closed states to determine amino acid positions at whichanalyte-binding dependent structural changes occur. In instances wherestructures for both open and closed states are not available, analysiscan be conducted by analogy to a structurally similar framework portion(e.g., PBP); (iv) includes cloning a signaling portion (e.g., a cpFP) atthe site identified in (iii) such that the analyte-binding dependentstructural change observed in (iii) will result in a conformationalchange in the signaling portion (e.g., the cpFP) and allostericmodulation of the signaling portion; (v) includes generating a libraryof mutants of biosensors with distinct linker sequences (e.g., by pointmutation), screening the library of mutants to identify mutants withenhanced properties (e.g., improved signal-to-noise ratio), andselecting mutants with enhanced properties (e.g., improvedsignal-to-noise ratio); (vi) includes increasing or decreasing bindingor affinity of the framework portion to the analyte, e.g., by modifyingamino acids in the interacting face of the framework portion or regionswithin the framework portion that are critical for analyte binding;(vii) includes increasing or decreasing signal emission by the signalingportion and/or changing the color of the signal where the signalingportion is a FP (e.g., a cpFP). Methods including (i)-(viii) areexemplified in the Examples section herein.

Methods of Use

The disclosure further provides methods for using the biosensorsdisclosed herein (e.g., one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186),including variants and/or fragments of the biosensor (e.g., variants orfragments of one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, 53, 62, 63, 77, 78, 91, 92, 93, 94, and/or 176-186)) to detectanalytes, e.g., in biological systems. Such methods can include, forexample:

Use of a maltose biosensor disclosed herein (e.g., one or more of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and/or 53 including variantsand/or fragments of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,and/or 53) to detect maltose, e.g., in a biological system;

Use of a glutamate biosensor disclosed herein (e.g., one or more of SEQID NOs: 62, 63, and/or 176-182 including variants and/or fragments ofSEQ ID NOs: 62, 63, and/or 176-182) to detect glutamate, e.g., in abiological system;

Use of a phosphonate biosensor disclosed herein (e.g., one or more ofSEQ ID NOs: 77 and/or 78 including variants and/or fragments of SEQ IDNOs: 77 and/or 78) to detect phosphonate, e.g., in a biological system;and/or

Use of a glucose biosensor disclosed herein (e.g., one or more of SEQ IDNOs: 91, 92, 93, 94, and/or 183 including variants and/or fragments ofSEQ ID NOs: 91, 92, 93, 94, and/or 183) to detect glucose, e.g., in abiological system.

Use of a GABA biosensor disclosed herein (e.g., SEQ ID NO: 184 includingvariants and/or fragments of SEQ ID NO: 184) to detect GABA, e.g., in abiological system.

Use of an ACh biosensor disclosed herein (e.g., one or more of SEQ IDNOs: 185 and/or 186 including variants and/or fragments of SEQ ID NOs:185 and/or 186) to detect ACh, e.g., in a biological system.

Techniques for performing such methods are known in the art and/or areexemplified herein. For example, methods can include introducing one ormore biosensors into a biological system (e.g., a cell); expressing theone or more biosensors in the biological system (e.g., the cell);monitoring the signal emitted by the expressed biosensor in thebiological system; and correlating the signal emitted by the expressedbiosensor in the biological system with a level of the analyte in thebiological system.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Maltose Indicators

Genetically encoded maltose indicators were generated using Escherichiacoli maltodextrin-binding protein (EcMBP) as a framework and eithercircularly permuted β-lactamase (cpBla) or circularly permutedfluorescent protein (cpFP) as a signal. Data describe below suggest thatcpBla and cpFP are not interchangeable.

Allosteric coupling of ligand binding to fluorescence was hypothesizedto require:

i) that the site in into which cpGFP is inserted have the capacity totransduce the global conformational change the scaffold protein (EcMBPin this example) to the local environment of the chromophore in cpGFP;and

ii) that the local environment of the chromophore (e.g., linkers) beoptimized to maximize the difference in emission between unbound (apo)and the bound (in this example maltose-bound) states.

Example 1A: Identification of cpGFP Insertion Sites in EcMBP

Potential insertion sites were identified using the crystal structuresof the maltose-bound, closed form of EcMBP (Ouiocho et al., Structure,5:997-1015, 1997) and the ligand-free, open form of EcMBP shown in FIG.1 (Sharff et al., Biochemistry, 31:10657-10663, 1992) to guide rationaldesign of EcMBP-cpGFP fusions that would result in maltose-dependent GFPfluorescence.

For (i), the change in dihedral angle (defined by the Cα atoms spanningfour residues) was analyzed to identify maltose-dependent structuralchanges in sequentially adjacent residues (FIG. 6); this analysis showedthat the Cα chain is “torqued” around residues 175 (ΔDihedral=)+41° and311 (ΔDihedral=)−22° upon ligand binding. This sequential conformationalchange was predicted to be coupled to structural changes of an insertedcpGFP, resulting in maltose-dependent fluorescence for the fusionprotein.

Previous studies using randomly digested and reassembled circularlypermuted β-lactamase (cpBla) and EcMBP showed maltose-dependentβ-lactamase activity in proteins with insertions of cpBla at EcMBPresidues 165 and 317 (Guntas et al., Chem. Biol., 11:1483-1487, 2004;Guntas and Ostermeier, J. Mol. Biol., 336:263-273, 2004).

Since the ΔDihedral of EcMBP165 is +11° (moderate change) and EcMBP317is +2° (no real change), four EcMBP-cpGFP templates were constructed byinserting cpGFP into EcMBP at sites 165, 175 (identified herein), 311(identified herein), and 317 to test our predictive method and theinterchangeability of cpBla and cpGFP at sites identified from theEcMBP-cpBla screen. These constructs were named MBP165-cpGFP,MBP175-cpGFP, MBP311-cpGFP, and MBP317-cpGFP (names were modified toencompass variants (e.g., with modified linker sequences). The cpGFPused is cpGFP146 described in Baird et al. (Proc. Natl. Acad. Sci., USA,96:11241-11246, 1999). PCR assembly was used to construct fusionproteins with GlyGly-linkers between EcMBP and each terminus of cpGFP.The amino acid sequence of each construct is shown in FIGS. 6-9. Thesequences of SEQ ID NOs:1-3 shown in FIGS. 7A-7C (i.e., MBP165-cpGFP)differ in the linker sequence between MBP 1-165 and cpGFP 147-238(linker 1: see the line ending in amino acid 240)). The sequences of SEQID NOs: 4-5 shown in FIGS. 8A-8B (i.e., MBP175-cpGFP) differ in thesequence between MBP 1-175 and cpGFP 147-238 (linker 1: see the lineending in amino acid 240)). The sequences of SEQ ID NOs: 6-7 shown inFIGS. 9A-9B (i.e., MBP311-cpGFP) differ in the sequence between cpGFP1-146 and MBP 312-370 (linker 2: see the line ending in amino acid640)). Each construct includes 3 linkers: A linker between theC-terminus of the C-terminal portion of MBP and the N-terminus of cpGFP(i.e., linker 2), a linker between the N-terminus of cpGFP andC-terminus of the N-terminal portion of MBP, and a linker in cpGFP(i.e., linker 3).

Example 1B: Linker Optimization

Libraries of variants of SEQ ID NOs: 1-8 were generated with randomizedlinkers by single-stranded uracil template mutagenesis (see Kunkel etal., Methods Enzymol., 204:125-139, 1991) using the primers listedbelow:

165 Linker 1 Primers: (SEQ ID NO: 9) PLIAADGxxNVYIM (SEQ ID NO: 10)PLIAADxxNVYIM (SEQ ID NO 11) PLIAADGGxxNVYIM (SEQ ID NO: 12)PLIAADGxPNVYIMG (SEQ ID NO: 13) PLIAADGIxNVYIMG (SEQ ID NO: 14)PLIAADPxSHNVYIM (SEQ ID NO: 15) PLIAADxPSHNVYIM (SEQ ID NO: 16)PLIAADxxSHNVYIM (SEQ ID NO: 17) PLIAADxxSHNVFIM (SEQ ID NO: 18)PLIAADPxSHNVFIM (SEQ ID NO: 19) PLIAADPxSYNVFIM (SEQ ID NO: 20)PLIAADxxSYNVFIM (SEQ ID NO: 21) PLIAADPxSYNVFIM (SEQ ID NO: 22)PLIAADxxSYNVFIM (SEQ ID NO: 23) PLIAADPxSxNVYIM (SEQ ID NO: 24)PLIAADPxSHxVYIM (SEQ ID NO: 25) PLIAADPxSHNxYIM (SEQ ID NO: 26)PLIAADPxSHNVxIM 165 Linker 2 Primers: (SEQ ID NO: 27) KLEYNFNxxYAFKYEN(SEQ ID NO: 28) KLEYNFNxYAFKYEN (SEQ ID NO: 29) KLEYNFNYAFKYEN(SEQ ID NO: 30) KLEYNFxYAFKYEN (SEQ ID NO: 31) KLEYNxxYAFKYEN(SEQ ID NO: 32) KLEYNWxYAFKYEN (SEQ ID NO: 33) KLEYNxKYAFKYEN(SEQ ID NO: 34) KLEYNFNPxYAFKYEN (SEQ ID NO: 35) KLEYNFNxPYAFKYEN175 Linker 1 Primers: (SEQ ID NO: 36) AFKYENxxSHNVYIM175 Linker 2 Primers: (SEQ ID NO: 37) KLEYNFNxxKYDIKDV311 Linker 1 Primers: (SEQ ID NO: 38) KSYEELxxSHNVYIM (SEQ ID NO: 39)KSYEELPxSHNVYIM (SEQ ID NO: 40) KSYEELxPSHNVYIM 311 Linker 2 Primers:(SEQ ID NO: 41) KLEYNFNxxAKDPRIA (SEQ ID NO: 42) KLEYNFNPxAKDPRIA(SEQ ID NO: 43) KLEYNFNxPAKDPRIA 317 Linker 1 Primers: (SEQ ID NO: 44)ELAKDPRxSHNVYIM (SEQ ID NO: 45) ELAKDPRxxSHNVYIM (SEQ ID NO: 46)ELAKDPRxxxSHNVYIM 317 Linker 2 Primers: (SEQ ID NO: 47) KLEYNFNxAATMENA(SEQ ID NO: 48) KLEYNFNxxAATMENA (SEQ ID NO: 49) KLEYNFNxxxAATMENA

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

About 400 variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM maltose.

Insertion of cpGFP as MBP317, a site previously reported for cpBla, didnot show maltose-dependent fluorescence (FIG. 11) even though theframework protein still bound maltose, as determined by isothermaltitration calorimetry (FIG. 12). These data demonstrate thatidentification of insertion sites by a method other than insertion ofcpGFP (such as insertion of cpBla) is not sufficient to identify sitesthat transduce ligand binding to changes in fluorescence intensity

Insertion of cpGFP at residue 165 of EcMBP (EcMBP165-cpGFP), anotherposition reported in cpBla studies (Guntas and Ostermeier, supra) with-GlyGly-linkers flanking the cpGFP resulted in a protein in whichfluorescence increased 20% (ΔF/F=0.2) upon addition of saturatingmaltose.

Screening a fully-degenerate, length-two library (“XX”) at either theEcMBP-cpGFP linker (linker 1) or the cpGFP-EcMBP linker (linker 2)yielded proteins with maltose-dependent fluorescent increases >300% ordecreases >50% (FIG. 11). Many of the variants with increased ΔF/Fvalues had linkers containing proline(s). Subsequent librariesconstructed from oligonucleotides encoding XP or PX and randomization ofthe residues in GFP from residue 146 to 150 were screened, yielding afinal variant with: a two-proline EcMBP-cpGFP linker, a two-glycinecpGFP-EcMBP linker, GFP-H148Y, and GFP-Y151F. This variant, called“EcMBP165-cpGFP.PPYF” (abbreviated PPYF (SEQ ID NO:2)) has a ΔF/F=2.5, aKd for maltose of 3 μM. Screens also identified variantEcMBP311-cpGFP.L2-NP (-AsnPro- at linker 2 (SEQ ID NO:7)), which has aΔF/F of 1.0 and a Kd for maltose of 2 μM. This variant has an inferiormaltose-dependent fluorescence increase than PPYF, but demonstratesgenerality of the cpFP insertion method.

EcMBP175-cpGFP was also screened with XX linkers, and a few variantswith ΔF/F≈1 were identified (FIG. 11). One mutant, with the first linkerencoding HL (EcMBP175-cpGFP.L1-HL (SEQ ID NO:5)), has a ΔF/F=0.5 and aKd for maltose of 1.3 μM.

These data support that choice of insertion site by structural analysisis preferable to random insertion.

Example 1C: Modifying Ligand Binding and/or Fluorescent Properties ofSensors

One objective in the development of generic biosensors is for theframework to permit independent optimization of binding and signalingproperties. Analysis of whether biosensors herein permit suchoptimization was tested using the high-SNR sensor PPYF, by: (i)rationally altering maltose-binding affinity; (ii) changing theligand-binding specificity from maltose to sucrose, and (iii) creating afamily of sensors in multiple colors.

As a first step, the impacts of mutations of three tryptophanside-chains in the maltose-binding pocket (W230, W62, and W340) weretested. These sites have previously been shown to lower the affinity ofEcMBP for maltose by one, two, or three orders of magnitude,respectively, when mutated to alanine (Martineau et al., J. Mol. Biol.,214:337-352, 1990). A mutation to the hinge region, I329W, was also madeto PPYF, as this has been shown to increase maltose affinity by about2-fold in both wild-type EcMBP (Marvin and Hellinga, Nat. Struc. Biol.,8:795-798, 2001) and in the EcMBP-cpBla switches (Guntas et al., Chem.Biol., 11:1483-1487, 2004; Kim and Ostermeier, Arch. Biochem. Biophys.,446:44-51, 2006). As shown in FIG. 13, for the PPYF sensor, the threetryptophan-to-alanine binding-pocket mutations behaved as expected,lowering affinity by between one and three orders of magnitude. Incontrast, the I329W mutation did not increase affinity as expected, butrather decreased it. ΔF/F also decreased. This data suggests that themechanism of fluorescence change in this sensor is dependent on subtleinteractions between EcMBP and cpGFP that are linked to the I329Wmutation.

As an alternative test for changing the ligand-binding specificity ofthe sensor while preserving fluorescence signaling, “5-7” mutations(D14L, K15F, W62Y, E111Y), previously shown to confer EcMBP with anaffinity for sucrose (Guntas and Mansell, Proc. Natl. Acad. Sci.,102:11224-11229, 2005), were made to PPYF. As shown in FIG. 14A, themutations conferred to the sensor about 2 mM affinity for sucrose and −3mM affinity for maltose. To address a discrepancy between expected(micromolar) and observed (millimolar) affinity for disaccharides, the5-7 mutations were made to sensors with cpGFP inserted at differentpositions in EcMBP, and with different linker compositions. In thecontext of EcMBP165-cpGFP.PCF, the 5-7 mutations conferred very low (butobservable) binding preference for sucrose over maltose (FIG. 14B). Thetrend of higher (but still weak) affinity for sucrose (˜0.6 mM) overmaltose (˜6 mM) continued when the 5-7 mutations are made in the contextof EcMBP175-cpGFP.L1-HL (FIG. 14C). In the context ofEcMBP311-cpGFP.L2-NP, the 5-7 mutations appeared to eliminate allbinding (FIG. 14D). The preference for sucrose over maltose of the 5-7variants of the sensors is consistent with the binding properties of the5-7 variants of EcMBP alone and EcMBP-cpBla (Guntas and Mansell, Proc.Natl. Acad. Sci., 102:11224-11229, 2005). The lower affinity for bothligands of the 5-7 variants of the sensors may be the consequence of theinserted cpGFP shifting the open and closed equilibrium.

These data suggest that ligand binding and fluorescent properties ofbiosensors can be independently modified.

Example 1D: Modifying Sensor Color

The color of GFP can be altered by changing the amino acids that eithercomprise or interact with the chromophore (see Shaner et al., J. Cell.Sci. 120:4247-4260, 2007, for a review).

Using PPYF as a template, mutations Y66W (to yield a cyan variant,“cpCFP”), L64F+T65G+V68L+T203Y (yellow, “cpYFP”), and Y66H (blue,“cpBFP”) mutations were made (see Cubitt et al., Trends Biochem.,20:448-455, 1995, for exemplary methods). As shown in FIG. 15, thevariants exhibit fluorescence emission spectra consistent with theirrespective intended designs.

The ΔF/F of the color variants in response to maltose is different (ineach case inferior) from the ΔF/F of 2.5 observed in PPYF-green. TheEcMBP165-cpYFP.PPYF sensor, which has the same covalent chromophorestructure as PPYF, has the greatest ΔF/F of the three spectral variants(FIG. 15A). EcMBP165-cpCFP.PPYF has a lower ΔF/F than the green andyellow variants, but by incorporating previously identified mutations,(L1-PC+GFP-Y151F; the resulting protein is called EcMBP165.cpCFP.PCF), avariant with ΔF/F=0.8 was obtained (FIG. 15A).

The EcMBP165-cpBFP.PPYF variant, while dimly fluorescent, is not asensor, and a screen of 800 linker variants failed to produce anyvariant with ΔF/F >0.2 (FIG. 16).

Since EcMBP165-cpBFP.PPYF was very dim, Azurite mutationsT65S+V150I+V224R were included to increase brightness and stability, andmake EcMBP165-cpAzurite a good template for linker screening. Usingoligonucleotides encoding XX amino acid linkers, a variant was obtained,EcMBP165-cpAzurite.L2-FE, that had ΔF/F=0.8 (FIG. 15).

Example 1E: Modifying Sensor Color and Ligand Specificity/Affinity

The four sucrose-binding “5-7” mutations described above that conferredweak sucrose affinity in the green sensor (EcMBP165-cpGFP.PPYF) wereconverted to blue, cyan, and yellow maltose sensors(EcMBP165-cpAzurite.L2-FE, EcMBP165-cpCFP.PCF, and EcMBP165-cpYFP.PPYF).The green and yellow sensors showed increased fluorescence upon additionof 10 mM sucrose, but the cyan and blue proteins did not (FIG. 15A).Like the green variant, the yellow variant had no detectable sucroseaffinity with the wild type binding pocket (FIG. 15C) and millimolaraffinity for both sugars, with preference for sucrose over maltose (FIG.15D).

As seen in FIG. 17, as maltose concentration increased, the blue sensorincreased in fluorescence first (Kd ˜2.7 μM), then the green (Kd ˜40μM), then the yellow (Kd ˜350 μM), and at high maltose concentrations,the cyan variant began to increase its fluorescence (Kd ˜1.7 mM).

Example 1F: Imaging Bacteria

The ultimate value of genetically encoded fluorescent sensors is intheir utility for observing analyte flux in living cells and organisms.In a simple proof-of-principle experiment, Escherichia coli expressingPPYF or PPYF.T203V (see “Second-generation maltose sensors” below) wereimaged in the green fluorescence channel in the absence of maltose, andthen re-imaged after addition of saturating maltose to the media.

As shown in FIG. 18, bacteria expressing the sensors clearly becamebrighter, while control bacteria expressing EGFP appeared unchanged.Increased fluorescence was quantified by measuring the peak (gray-value)pixel intensity of each bacterium. Those expressing PPYF undergo anapproximate doubling of fluorescence (bacterium-averaged ΔF/F=1.1±0.4),those expressing PPYF.T203V have slightly increased ΔF/F(ΔF/F=1.29±0.2), while those expressing EGFP have no change influorescence (ΔF/F=−0.01±0.05).

Example 1F: 2-Photon Imaging of Mammalian Cells

Multi-photon microscopy opened new frontiers for in vivo fluorescenceimaging, in particular for neuronal activity visualization through theuse of genetically encoded calcium indicators (Tian et al., Nat.Methods, 3:281-286, 2009; Denk et al., Science, 248:73-76, 1990; Denkand Svoboda, Neuron, 18:351-357, 1997).

To demonstrate that the maltose sensors described herein have thepotential to be used for 2-photon imaging, fluorescence excitationspectra were collected. As shown in FIG. 19, with a 535 nm bandpassemission filter (50 nm bp), EcMBP165-cpGFP.PPYF showed a 10-foldmaltose-dependent increase in fluorescence when excited at 940 nm. Allfour spectral variants showed a significant maltose-dependent increasein 2-photon fluorescence.

Example 1G: Sub-Cloning Maltose Sensors

EcMBP165-cpGFP.PPYF.T203V (see “Second-generation maltose sensors”below) were cloned into a modified version of the pDisplay vector(Invitrogen) for extracellular display on the surface of transientlytransfected human embryonic kidney (HEK293) cells.

As shown in FIG. 20, the sensor localized to the plasma membrane andincreased in brightness in a concentration-dependent manner whenperfused with buffers of varying maltose concentration. The ΔF/F is5.8-fold, very close to that of the soluble protein produced in E. coli,with the mid-point of the maltose-dependent fluorescence increase being6.5 μM (FIG. 21A), very similar to the affinity determined on purifiedprotein (5 μM). Furthermore, the surface displayed sensor respondedrapidly to a pulse of 1 mM maltose (FIG. 21A), indicating that the timecourse for its action is useful for transient events.

Example 1H: Crystal Structure Analysis of Maltose Sensors

High-resolution structures of several of the maltose sensors describedabove were generated. Crystallization trials were performed withEcMBP165-cpGFP.PPYF, EcMBP175-cpGFP.L1-HL, and EcMBP311-cpGFP.L2-NP inthe presence and absence of excess maltose, from which bothEcMBP175-cpGFP.L1-HL and EcMBP311-cpGFP.L2-NP crystallized in thepresence of maltose. X-ray structures were solved to 1.9 and 2.0 Åresolution, respectively, by molecular replacement (FIGS. 22A-22C).

The structures of the cpGFP and EcMBP domains of the sensors aresuperimposable with published crystal structures of cpGFP (from GCaMP2;RMSD=0.36 and 0.38 Å, respectively, for comparing 221 common Cα atoms)and EcMBP-maltose (RMSD=0.43 and 0.37 Δ, 370 Cα). The structure of EcMBPis largely unperturbed by insertion of the cpGFP domain; only residuesaround the 175 and 311 insertion sites showed any significantdisplacement.

GFP-H148, which H-bonds the GFP chromophore in the structure of nativeGFP, also directly H-bonded to the chromophore in theEcMBP175-cpGFP.L1-HL-maltose structure (FIG. 22B), although a differentrotamer was observed. In the EcMBP311-cpGFP.L2-NP-maltose structure,GFP-H148 is pulled away from the chromophore and is largely replaced bythe Asn from linker 2, which makes H-bond interactions to both strand 8of the GFP barrel and the chromophore phenolate oxygen (through a watermolecule, FIG. 22D). GFP-H148, meanwhile, seemed to stabilize theconformation of linker 2 of EcMBP311-cpGFP.L2-NP by H-bonding thebackbone carbonyl of the linker 2 Asn. There is some solvent access tothe cpGFP chromophore through the hole in the GFP barrel created bycircular permutation, although the inter-domain linkers block much ofthe opening in both structures. Relatively few contacts are made betweenthe cpGFP and EcMBP domains.

Based on the structures of two maltose-bound sensors, the sensingmechanism likely involves a shift in the relative position of linker 1and linker 2 induced by the conformational change in the EcMBP domainassociated with maltose binding (FIG. 5). The register shift ofinteractions between the two linkers could alter the proximity of linker2 and nearby side-chains to the cpGFP chromophore and change the waterstructure in the cpGFP opening, leading to a shift in the chromophoreprotonation equilibrium. This might explain why rigid proline ispreferred in either linker, since conformational changes upon ligandbinding might be better propagated through the rigid linkers to thecpGFP chromophore environment.

Example 1I: Generation of Second-Generation Maltose Sensors

In an attempt to increase brightness and ΔF/F of GCaMP, the localenvironment of the chromophore was altered by randomizing residueswithin cpGFP, and screening for improved variants (Tian et al., nat.Methods, 6:875-881, 2009).

As shown in FIG. 23, in the context of EcMBP165-cpGFP.PPYF, the T203Vmutation decreases the fluorescence emission of the apo-state by half(FIG. 23A), while saturated fluorescence and affinity are unchanged(FIG. 23B), increasing ΔF/F to 6.5. In the maltose-saturated state, PPYFitself has about a quarter the brightness of EGFP, and half thebrightness of cpGFP.

In the context of EcMBP311-cpGFP.L2-NP, the T203V mutation decreases thebrightness of both the apo-state and the saturated-state equally,resulting in no significant change in ΔF/F (FIGS. 23C and D).

These results indicate that the benefits of the T203V mutation are notuniversally transferable, and that cpGFP-based fluorescent sensors needto be optimized individually.

Example 2: Maltotriose Indicators

Genetically encoded maltotriose indicators were created using Pyrococcusfuriosus maltotriose binding protein. As described below, only thestructure of the ligand-bound state P. furiosus maltotriose bindingprotein (PfMBP) is available. As shown in FIGS. 1 and 2, PfMBP ishomologous to EcMBP (compare FIGS. 1 and 2). Two sensors were made,PfMBP171 and PfMBP316, the insertion points for which were selectedbased on homology to EcMBP165 and EcMBP311, respectively. Linkers wereoptimized. PfMBP sensors have a ΔF/F of ˜1.2.

Pyrococcus furiosus is a thermophilic organism. Proteins fromthermophiles have been shown to be more amenable to mutation than thosefrom mesophiles (Bloom et al., Proc. Natl. Acad. Sci., 103:5869-5874,2006). As an alternative to developing new sensors by inserting cpGFPinto PBPs, it should also be possible to generate new sensors bychanging the ligand-binding specificity of an existing PBP-based sensor.

It has previously been shown that the binding sites of PBPs can bereengineered to accommodate novel ligands (Looger et al., Nature,423:185-190, 2003). However, those re-design efforts used frameworkproteins from mesophiles and suffered from poor stability. Wehypothesized that PfMBP, which is intrinsically more stable than EcMBP,is more tolerant of mutations. To test this hypothesis, we characterizedand compared the stability of PfMBP to EcMBP, PfMBP-cpGFP sensors toEcMBP-cpGFP sensors, PfMBP binding site mutants to EcMBP binding sitemutants, and PfMBP-cpGFP sensor binding site mutants to EcMBP-cpGFPsensor binding site mutants. Conclusively, the PfMBP variants were morestable than the EcMBP variants. Finally, we demonstrate that theincreased thermo-stability of the PfMBP-cpGFP sensors is useful for themeasurement of maltotriose at temperatures as high at 60° C., whereasthe EcMBP-cpGFP sensors are only useful for the measurement of maltoseat temperatures as high as 40° C.

Example 2A: Identification of cpGFP Insertion Sites in PfMBP

The ligand-bound (closed) structure of PfMBP is available (Evdokimov etal., J. Mol. Biol., 305:891-904, 2001), but the unbound structure isnot. Accordingly, insertion sites for the PfMBP-cpGFP sensors wereidentified by homology to EcMBP.

Sites were selected based on the structural similarities between PfMBPand EcMBP. Two sites were selected. One of these sites is EcMBP311,which is homologous to PfMBP316. This site is at juncture between theend of the cluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). Another site that was made into asensor in EcMBP was EcMBP165, which is homologous to PfMBP171. cpGFP wasinserted into PfMBP at each of these sites. The sequences of theresulting constructs, PfMBP171-cpGFP and PfMBP316-cpGFP, are shown inFIGS. 24 and 25, respectively.

Example 2B: Linker Optimization

Libraries of variants of SEQ ID NOs: 50-53 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

175 Linker 1 Primers: (SEQ ID NO: 54) AIAQAFxxSHNVYIMA (SEQ ID NO: 55)AIAQAFPxSHNVYIMA 171 Linker 2 Primers: (SEQ ID NO: 56) KLEYNFNxxYYFDDKTE316 Linker1 Primers (SEQ ID NO: 57) VLDDPExxHNVYIM (SEQ ID NO: 58)VLDDPEIxxSHNVYIM 316 Linker2 Primers (SEQ ID NO: 59) KLEYNFxxNDPVIY(SEQ ID NO: 60) KLEYNFNxPKNDPVIY (SEQ ID NO: 61) KLEYNFNPxKNDPVIY

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 1 mM maltotriose.

Screening a fully-degenerate, length-two library (“XX”) at either thePfMBP171-cpGFP linker (linker 1) or the cpGFP-PfMBP linker (linker 2)yielded proteins with maltotriose-dependent fluorescent increases >100%or decreases >20% (FIG. 26A). A variant from this group with a GlyGlyPfMBP-cpGFP linker and a PheGlu cpGFP-PfMBP linker was selected forfurther characterization. This variant, called “PfMBP171-cpGFP.L2FE” hasa ΔF/F=1.2, a Kd for maltotriose of <1 μM.

Screening a fully-degenerate, length-two library (“XX”) at either thePfMBP316-cpGFP linker (linker 1) or the cpGFP-PfMBP linker (linker 2)also yielded proteins with maltotriose-dependent fluorescentincreases >100% or decreases >20% (FIG. 26B). A variant from this groupwith a GlyGly PfMBP-cpGFP linker and a PheGlu cpGFP-PfMBP linker wasselected for further characterization. This variant, called“PfMBP316-cpGFP.L1-NP” has a ΔF/F=1.2, a Kd for maltotriose of 40 μM.

These data support that structurally homologous frameworks can becompared to identify insertion sites for cpGFP.

Example 2C: Characterization of the Thermostability of the PfMBP andPfMBP-cpGFP Compared to EcMBP and EcMBP-cpGFP

Thermal stability of PfMBP171-cpGFP.L2FE was measured usingcircular-dichroism (CD) and compared to the original EcMBP and PfMBPbinding proteins, along with cpGFP. Following the changes by means of CDallowed determination of whether different transitions happened inalpha, beta, or both kinds of structures.

Given that cpGFP is a beta barrel, strong transitions in the beta signalalone were associated with changes in this kind of structure. In thesame way, transitions in both kinds of signals were associated with thebinding protein structure. As shown in FIG. 27A, PfMBP is significantlymore thermo-stable than EcMBP. In fact, while EcMBP denatured at about50° C., PfMBP did not denature at temperatures less than 80° C. Also,the addition of maltose to EcMBP stabilized the protein by about 10° C.

As shown in FIG. 27B, the stability of the EcMBP component of theEcMBP165-cpGFP.PPYF sensor decreased from 50° C. to 45° C. withinsertion of cpGFP, while the intrinsic stability of cpGFP in the sensorremained unchanged. There was little change in the stability of thePfMBP component of the PfMBP171-cpGFP.L2FE sensor with insertion ofcpGFP (FIG. 27B). Moreover, PfMBP seemed to exert a small stabilizingeffect over the inserted cpGFP, as shown by the change in the steepnessand melting point of the curve of the soluble form and thePfMBP171-cpGFP.L2FE sensor. All the associations made betweentransitions and domain unfolding were supported by CD spectra taken atthe beginning and the end of each temperature ramp.

Analysis of whether the PfMBP scaffold was more tolerant of mutationthan the EcMBP scaffold was also performed. Proof-of-principle mutationswere made to the ligand-binding sites of EcMBP and PfMBP, and theirrespective sensors. In EcMBP, Asn12 was mutated to Trp to result insteric clashes with the surrounding residues, and backbone, of thebinding pocket. The homologous mutation in PfMBP is Ala13Trp, whichwould be expected to have the same effect.

As shown in FIG. 27C, N12W decreased the Tm of EcMBP from 50° C. to 40°C., while the corresponding mutation in PfMBP, A13W, had no noticeableeffect. This data confirms that the thermo-philic protein is moretolerant of mutations to the binding site. Furthermore, in the contextof the sensors, the N12W mutation to EcMBP165-cpGFP.PPYF completelydestabilized the binding protein component of the sensor (FIG. 27D),while the A13W mutation in PfMBP171-cpGFP.L2FE had no effect onstability (FIG. 27D).

Example 2D: Tolerance of PfMBP Sensor to Increased Temperature

Fluorescence of the protein in the apo and ligand-bound states at wasmeasured at different temperatures.

As shown in FIG. 28A, fluorescence of the EcMBP165-cpGFP.PPYF sensor inthe bound state was higher than it is in the apo-state at lowertemperatures, by about 4-fold. However, at around 55° C. (the unfoldingtransition of the EcMBP component) the fluorescence of theEcMBP165-cpGFP.PPYF sensor dropped precipitously. As a result,EcMBP165-cpGFP.PPYF is unsuitable for detection of maltose attemperatures greater than 50° C. (FIG. 28B). In contrast,PfMBP171-cpGFP.L2FE retained its maltotriose binding capabilities athigh temperatures (FIGS. 28A and 28B), and is limited only by theintrinsic fluorescence of the cpGFP component, which decays at about 80°C. (FIG. 28A).

Example 2E: Measurement of Maltodextrins in Hot Liquids

To demonstrate that the soluble and immobilized sensors functionsimilarly, PfMBP171-cpGFP.L2FE, PfMBP316-cpGFPL1XXX, andEcMBP165-cpGFP.PPYF.T203V were immobilized via their N-terminalpoly-histidine tags on to the surface of Ni-NTA coated glass. In afluorescence plate reader, the immobilized proteins performed similarlyto their soluble counterparts (see FIGS. 28C, 28D, and 28F).

Next, a prototype device was constructed, with a light guide providingthe excitation light and returning the fluorescent emitted light back tothe photodetector, the bio-sensor protein immobilized to Ni-NTA coatedcoverslips, and the coverslip attached to the end of the light guide.The “wand” of the detector was dipped into different compositions ofsolutions, each with varying concentrations of maltose or maltotriose.Experiments were performed at different temperatures. PfMBP-cpGFP sensorperformed better at higher temperatures (as high as 60° C.) than theEcMBP-cpGFP sensor.

Example 3: Glutamate Indicators

Glutamate indicators were created from Escherichia coliglutamate-binding protein (EcYbeJ). As with PfMBP in Example 2, only thestructure of the ligand-bound EcYbeJ is available. EcYbeJ is homologousto EcMBP, but to a lesser degree. The best homology match between a sitein EcYbeJ and a site in a binding protein for which an intensity-basedsensor has already been created is EcYbeJ253 and EcMBP311 (describedherein). As shown in FIG. 3, both sites are at the junction of “RisingHelix 8” and the “Equatorial Helix/Coil.” The amino acid composition ofthe cpGFP and EcYbeJ junction was made the same as that of theEcMBP311-cpGFP sensor (Linker 2=NP). The amino acid composition of theEcYbeJ junction and cpGFP was optimized to LV (Linker 1=LV). The varianthas a ΔF/F of 5.

Example 3A: Identification of cpGFP Insertion Sites

The ligand-bound (closed) structure of Shigella flexneri glutamatebinding protein is available (Fan et al., Protein Pept. Lett.,13:513-516, 2006). This protein has only 4 amino acid mutations relativeto EcYbeJ, and is thus an appropriate model.

Insertion sites for the EcYbeJ-cpGFP sensors were identified by homologyto EcMBP. Based on the topology map (FIG. 3), position 311 in EcMBP wasidentified as an acceptable insertion site for EcYbeJ. EcMBP311 isequivalent to EcYbeJ253. EcYbeJ253 is at juncture between the end of thecluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). In YbeJ, the structure that ishomologous to the equatorial helix is the equatorial coil (depicted inred, to match the red coloring of Helix 9).

Intrinsic affinity of wild-type YbeJ for glutamate (˜1 μM) was too highto permit high-throughput screening of linker libraries. Endogenousglutamate (from the growth media) saturates the sensor, makingmeasurement of the unbound state technically challenging. A mutation toYbeJ (A184V), in the “hinge” of the protein were made. Mutation of thisresidue to Trp or Arg have previously been shown to decrease affinity inFRET-based sensors (see Okumoto et al., Proc. Natl. Acad. Sci.,102:8740-8745, 2005). EcYbeJ253 (A184V)-cpGFP has an affinity forglutamate of about 100 μM. All references to EcYbeJ253-cpGFP, unlessotherwise noted, refer to the A184V variant. The sequences of the EcYbeJconstructs are shown in FIG. 29.

Example 3B: Linker Optimization

Libraries of variants of SEQ ID NOs: 62-63 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

253 Linker 1 Primers: (SEQ ID NO: 64) FKNPIPPxSHNVYIMA (SEQ ID NO: 65)FKNPIPPxxSHNVYIMA (SEQ ID NO: 66) FKNPIPPPxSHNVYIMA (SEQ ID NO: 67)FKNPIPPxPSHNVYIMA (SEQ ID NO: 68) KWFKNPIxxSHNVYIMA (SEQ ID NO: 69)FKNPIPPxxNVYIMAD (SEQ ID NO: 70) KWFKNPIxxNVYIMAD 253 Linker 2 Primers:(SEQ ID NO: 71) KLEYNFNxKNLNMNF (SEQ ID NO: 72) KLEYNFNxxKNLNMNF(SEQ ID NO: 73) KLEYNFNxPKNLNMNF (SEQ ID NO: 74) KLEYNFNPxKNLNMNF(SEQ ID NO: 75) GHKLEYNxxLNMNF (SEQ ID NO: 76) KLEYNFNxxLNMNF

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM glutamate.

Screening a fully-degenerate, length-two library (“XX”) at theEcYbeJ253-cpGFP linker (linker 1) identified a sensor withglutamate-dependent fluorescent increases of ˜100%. This variant has aLeuVal EcYbeJ-cpGFP linker (L1-LV) and was used as the framework foroptimization of the cpGFP-EcYbeJ253 linker (linker 2). The results ofthat screen yielded a protein with glutamate-dependent fluorescentincrease of ˜500% and a linker 2 composition of AsnPro. As shown in FIG.30, this variant, called “EcYbeJ253-cpGFP.L1LVL2NP” has a ΔF/F=5, a Kdfor glutamate of 100 μM. Interestingly, the composition of the secondlinker, AsnPro, is the same as the linker composition ofEcMBP311-cpGFP.L2NP.

Example 3C: Detection of Extracellular Glutamate

EcYbeJ253-cpGFP.L1LVL2NP was cloned into the pDisplay™ vector to allowtargeting and anchoring of the sensor to the plasma membrane. Theresulting construct was transfected into cultured mammalian cells(HEK293) to visualize the addition of glutamate to extracellular media.Constructs were also generated in a bacterial expression vector with theepitope tags individually and in combination.

As shown in FIG. 31, the hemagglutinin tag interferes with thefluorescence change. EcYbeJ253-cpGFP.L1LVL2NP was re-cloned into aderivative of the pDisplay™ vector, lacking the hemagglutinin tag,called pMinDis (for Minimal Display). This new construct, when expressedin HEK293 cells, shows a change in fluorescence intensity under 2-photonexcitation that is approximately the same as the soluble protein (seeFIG. 32) with higher affinity, of about 1 μM (see FIG. 32).

To demonstrate that the sensor is functional in neurons, and not justcultured HEK cells, the gene from EcYbeJ253-cpGFP.L1LVL2NP was clonedinto an adeno-associated virus vector (AAV) under control of thesynapsin promoter. Virus particles were generated and used to infectcultured primary hippocampus neurons from rats 7 days after culturing.14 days after culturing (and 7 days after infection), the infectedneurons were imaged under 2-photon microscopy (FIG. 33).

Example 4: Phosphonate Indicators

An indicator for phosphonate compounds was created from Escherichia coliphosphonate-binding protein (EcPhnD). In this instance, only thestructure of the ligand-bound state was available at the time the sensorwas conceived. EcPhnD is homologous to EcMBP to a lesser degree and toEcYbeJ to a greater degree. The best homology match between a site inEcPhnD and a site in a binding protein for which an intensity-basedsensor has already been created is EcPhnD90 and EcYbeJ253. There is no“Rising Helix 8” in EcPhnD, but there is an “Equatorial Helix/Coil”(FIG. 4). cpGFP was inserted at the Equatorial Helix/Coil and linkerswere optimized to yield a sensor with ΔF/F of 1.2. EcPhnD is a dimmer,so, a pair of mutations (L297R+L301R) were made to convert it to amonomer. The monomer variant has a ΔF/F of 1.6.

Example 4A: Identification of cpGFP Insertion Sites in EcPhnD

Insertion sites for the EcPhnD-cpGFP sensors were identified using theligand-bound (closed) structure of EcPhnD by homology to EcMBP. Based onthe topology map (FIG. 4), position 311 in EcMBP was identified as anacceptable insertion site in EcPhnD. EcMBP311 corresponds to EcPhnD90.This site is at the point where the rising strand (Strand D) of EcPhnDhas a small bend in it that runs equatorial to the rest of the sheets inthe protein. Even though it is topologically different from the“equatorial” spanning helix (Helix 9) of EcMBP its equatorial alignmentis similar, and with just the closed structure at the time, in anenvironment that was expected to undergo significant dihedral changeupon binding ligand. Sequences of EcPhnD constructs are shown in FIG.34.

Example 4B: Linker Optimization

Libraries of variants of SEQ ID NOs: 77-78 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

90 Linker 1 Primers: (SEQ ID NO: 79) QTVAADGSSHNVYIMA (SEQ ID NO: 80)QTVAADxxSHNVYIMA (SEQ ID NO: 81) QTVAADxPSHNVYIMA (SEQ ID NO: 82)QTVAADPxSHNVYIMA (SEQ ID NO: 83) QTVAADxxNVYIMA (SEQ ID NO: 84)QTVAADxxSHNVYIMA (SEQ ID NO: 85) VFQTVAxxSHNVYIMA 90 Linker 2 Primers:(SEQ ID NO: 86) HKLEYNFNPGYWSVLI (SEQ ID NO: 87) HKLEYNFNxxPGYWSVLI(SEQ ID NO: 88) HKLEYNxxPGYWSVLI (SEQ ID NO: 89) HKLEYNFNxxYWSVLI(SEQ ID NO: 90) HKLEYNFNPxYWSVLI

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 100 uM 2AEP.

Screening a number of fully-degenerate, libraries at the EcPhnD90-cpGFPlinker (linker 1) yielded a protein with 2AEP-dependent fluorescentincreases of >100%. This variant has a AlaAsp EcPhnD-cpGFP linker(L1-AD) and a ΔF/F of 1.2. The variant came from a linker that alsodeleted two residues, effectively making the insertion point of cpGFPoccur after residue D88, and then skipping to residue P91 at thecpGFP-EcPhnD linker.

It was observed from the crystal structure that EcPhnD forms a dimer. Todisrupt the dimer inter-face and potentially simplify the observablebinding behavior of the EcPhnD protein, two mutations, L297R and L301R,were introduced into the dimerization helices. These mutations wereexpected, by charge repulsion, to disrupt the dimer interface. As shownin FIG. 35, incorporation of L279R and L301R mutations intoEcPhnD90-cpGFP.L1AD caused ΔF/F to increases to 1.6 in response to 2AEP.

Further attempts to crystallize the open, ligand-unbound form of theprotein were successful after making a mutation to the binding site,H157A, that substantially decreased affinity for phosphonate compounds.This mutant was crystallized in the absence of ligand, and the openstate of the protein solved. The ΔDihedral analysis (FIG. 36) showedthat the region of greatest dihedral change was the group of residuesfrom 88-90, just one amino acid away from the site chosen by homology tothe equatorial helix.

These data further indicate that ΔDihedral metric is sufficient foridentifying sites in PBPs into which cpGFP can be inserted and result inintensity-based fluorescent sensors.

Example 5: Glucose Indicators

Glucose indicators were created from Thermus thermophilus glucosebinding protein (TtGBP). In this instance, only the structure of theligand-bound state is available. TtGBP is very homologous to EcMBP andPfMBP (compare FIG. 5 with FIGS. 1 and 2). The insertion point(TtGBP326) was chosen by homology to EcMBP311 and PfMBP316. The aminoacid composition of the cpGFP and TtGBP junction was made the same asthat of the EcMBP311-cpGFP and EcYbeJ253 sensors (Linker 2=NP). Linker 1was optimized (Linker 1=PA) and the TtGBP326 sensor have a ΔF/F of ˜2.5.To improve its utility for the measuring glucose concentrations in humanblood, the affinity was weakened from its native ˜1 μM to 1.5 mM bymutation of two residues in the binding pocket (H66A+H348A).

Example 5A: Identification of cpGFP Insertion Sites in TtGBP

The ligand-bound (closed) structure of TtGBP is available (Cuneo et al.,J. Mol. Biol., 362:259-270, 2006). Accordingly, insertion sites for theTtGBP-cpGFP sensors were identified by homology to EcMBP and PfMBP.Based on the topology map (FIG. 5), it is apparent that TtGBP, PfMBP,and EcMBP are structurally similar in the closed, ligand-bound state.Positions in EcMBP determined by the dihedral analysis (see above) werepredicted to be acceptable insertion sites in TtGBP. EcMBP311 ishomologous to TtGBP326. This site is at juncture between the end of thecluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). The amino acid sequence of theTtGBP construct is shown in FIG. 37.

Example 5B: Linker Optimization

Libraries of variants of SEQ ID NO:91 were generated with randomizedlinkers by single-stranded uracil template mutagenesis using the primerslisted below:

326 Linker 1 Primers: (SEQ ID NO: 95) DSDPSKYxxSHNVYIM (SEQ ID NO: 96)DSDPSKYPxSHNVYIM (SEQ ID NO: 97) DSDPSKYxPSHNVYIM (SEQ ID NO: 98)RLDSDPSxxSHNVYIM (SEQ ID NO: 99) DSDPSKYxxNVYIM 326 Linker 2 Primers:(SEQ ID NO: 100) KLEYNFNxxNAYGQSA (SEQ ID NO: 101) KLEYNFxxPNAYGQSA(SEQ ID NO: 102) GHKLEYNxxNAYGQSA (SEQ ID NO: 103) KLEYNFNxPNAYGQSA(SEQ ID NO: 104) KLEYNFNPxNAYGQSA

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several hundred variants were screen in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM glucose.

Linker 1 was optimized (Linker 1=PA) and the TtGBP326-cpGFP.L1PAL2NPsensor has a ΔF/F of ˜2.5 (see FIG. 38). Additionally, the TtGBP sensorwas tested with and without the N-terminal pRSET tag and no differencewas observed. Specifically, both sensors exhibited an affinity forglucose of about 1.5 mM and a ΔF/F of 2.5.

Data showing that it was possible to construct a glucose sensor byreplacing the EcMBP or PfMBP with TtGBP, retaining the composition oflinker 2, and optimizing the composition of linker 1, indicates that themethods for generating sensors disclosed herein can be used to generatesensors using any suitable framework.

Example 5C: Detecting Changes in Glucose Concentration in Vivo

The TtGBP326-cpGFP.L1PAL2NP sensor was cloned into a variant of thepDisplay™ vector lacking the N-terminal secretion sequence, theN-terminal hemagglutinin tag, the C-terminal cMyc tag, and theC-terminal PDGFR membrane anchoring domain.

The TtGBP sensor was cloned into a mammalian expression vector (based onthe pDisplay™ vector described in Example 3 above) with the secretion,epitope, and transmembrane anchoring peptides removed, thus resulting incytosolic expression of the TtGBP326-cpGFP.L1PAL2NP+H66A+H348A sensor.The construct was transfected into HEK293 cells. As shown in FIG. 39,the TtGBP sensor was expressed in the cytosol.

As shown in FIG. 40, addition of 10 mM glucose to the media increasesfluorescence.

The TtGBP326-cpGFP.L1PAL2NP+H66A+H348A sensor was further modified byL276V mutation to produce TtGBP326.L1PA.L2NP.H66A.H348A.L276V (see FIG.50). As shown in FIG. 51, this construct has an affinity for glucose of6.5 mM.

Additionally, the TtGBP326.L1P1.L2NP.G66A.H348A.L276V was cloned intothe pMinDis derivative of the pDisplay vector and expressed on theextracellular surface of HEK293 cells. After exchanging the HEK293 cellmedia for PBS, addition of glucose to the PBS led to an increase influorescence (see FIG. 52).

These data indicate, in part, that the pRSET tag is not essential to thefunction of the sensor and that the TtGBP326-cpGFP.L1PAL2NP sensor iscapable of detecting changes in the concentration of glucose inside oron the external surface of human cells.

Example 6: Stability, Affinity and Chromatic Variants of the GlutamateSensor iGluSnFR Example 6A: In Vivo Assessment of iGluSnFR Brightness inApical Dendrites in Mouse Somatosensory Cortex

Wildtype C57/B6 mice were purchased from the Jackson Laboratory andgroup housed in the Janelia animal facility. Mice were injected at 8weeks of age with AAV2/1.hSynapsin1.iGluSnFR.A184S or SF-iGluSnFR.A184S,at identical titers (1×10¹³ genomic copies per milliliter, GC/ml),volumes (20 nl), and locations (3 mm lateral to midline, 1.4 mm caudalto bregma, and 0.3 mm below the cortical surface). After viralinjection, a craniotomy (3 mm diameter) was made over the injectionsite, and the skull was replaced with a #1.5 Schott glass and fixed inplace with dental acrylic (Lang Dental Manufacturing), which alsosecured a titanium head bar to the skull for head-mounting duringimaging experiments.

In vivo two-photon imaging experiments were performed during a state of‘quite wakefulness’, after having been habituated to head fixation theprior 2-3 days. Period water rewards were given to keep animals hydratedand passive. For comparisons of intensity and bleaching, a customtwo-photon microscope emitting 960 nm light from a Coherent Chameleonultrafast laser was used. All experiments were performed using a 25×,1.5 NA Olympus objective immersed in water. Image acquisition wasperformed with Scanlmage (Vidrio) software and analyzed post hoc usingImageJ (NIH). Images were acquired at a variety of speeds/zooms, andpowers in order to assess the impact of pulse energy and dwell time onbleaching and intensity. Images at each setting were acquired for 5seconds. To analyze the data, images were averaged and thresholded tocreate a signal (above threshold) and background mask. Signals in thesemasks were then averaged, and SNR was calculated from these as(signal-background)/(standard deviation of background). Bleachingpercentage was calculated as the average intensity in the first 25% ofthe trace, divided by the last 25% of the trace.

Example 6B: Ferret Visual Cortex Assessment of SF-iGluSnFR.A184S andA184V

All procedures were approved by the Max Planck Florida Institute forNeuroscience Institutional Animal Care and Use Committee and adhered tothe standards of the National Institutes of Health. Juvenile femaleferrets (Mustela putorius furo, Marshall Farms) were used. Animals werehoused in a vivarium under 16 hour light/8 hour dark cycle. The fullmethodological details for functional two-photon imaging of ferretvisual cortex is previous described in detail (Wilson et al., 2016, Nat.Neurosci., 19:1003-9).

Briefly, juvenile female ferrets (Mustela putorius furo, Marshall Farms)aged P21-22 (n=2) were anesthetized with ketamine (50 mg/kg, IM) andisoflurane (1-3%) delivered in O₂, then intubated and artificiallyrespirated. Atropine (0.2 mg/kg, SC) and a 1:1 mixture of lidocaine andbupivacaine administered subcutaneously in the scalp. Animals were keptat 37° C. A small craniotomy (0.8 mm) was made over the visual cortex7-8 mm lateral and 2-3 mm anterior to lambda. AAV2/1.hSynapsin1.Cre(Penn Vector Core) was diluted in phosphate-buffered saline (Sigma) andmixed with AAV2/1.hSynapsin-FLEX.SF-iGluSnFR.A184S or A184V forexpression in layer 2/3 cortical neurons. Beveled glass micropipetteswere lowered into the brain and 400-500 nl of virus were injected over 5minutes at multiple depths below the pia. Following, the craniotomy wasfilled with 1% w/v agarose.

After four weeks, ferrets were anesthetized with 50 mg/kg ketamine and1-3% isoflurane. Atropine (0.2 mg/kg, SQ) and bupivacaine wereadministered. Animals were kept at 37 to 38° C., artificiallyrespirated, and given intravenous fluids. Isoflurane (1-2%) was usedthroughout the surgical procedure to maintain a surgical plane ofanesthesia. ECG, endtidal CO₂, external temperature, and internaltemperature were continuously monitored. A custom titanium headplate wasimplanted on the skull at the viral injection site and the duraretracted to reveal the cortex. A custom insert with a single 4 mmcoverglass (0.17 mm thickness) was placed onto the brain to gentlycompress the underlying cortex and dampen biological motion duringimaging. The cranial window was hermetically sealed using a stainlesssteel retaining ring and Vetbond. Tropicamide Ophthalmic Solution andPhenylephrine Hydrochloride Ophthalmic Solution were applied and contactlenses were inserted into both eyes. Upon completion of the surgicalprocedure, Isoflurane was gradually reduced and pancuronium (2mg/kg/hour) was delivered IV to immobilize the animal.

The animal was placed under the microscope 25 cm from the stimulusmonitor, with the monitor subtending 130 degrees in azimuth and 74degrees in elevation. Imaging was performed using a Bergamo II(Thorlabs) running Scanlmage 5 or Scanlmage 2015¹⁹ (Vidrio Technologies)with dispersion compensated 950 nm excitation provided by an Insight DS+(Spectraphysics). Average excitation power after the exit pupil of theobjective (16×, CFI75, Nikon Instruments) ranged from 25 to 40 mW.Two-photon frame triggers from Scanlmage were synchronized with stimulusinformation using Spike2 (CED). Visual stimuli were generated usingPsychoPy (Peirce, 2007, J. Neurosci. Methods, 162:8-13). Full-fielddrifting square-wave gratings (16 directions, 100% contrast, 0.1cycles/°, 4 cycles/sec., 3 sec. stimulus period followed by 2-3 sec.ISI, plus a blank) were presented to the contralateral eye in apseudorandom sequence for 8 trials.

Images were corrected for in-plane motion using a correlation-basedapproach (MATLAB). ROI drawing was performed in ImageJ (Schindelin etal., 2012, Nat. Methods, 9:676-82). Fluorescence time-courses werecomputed as the mean of all pixels within the ROI at each time point andwere extracted as described in Sage et al. (2012, ImageJ User developerConference 1:1). Fluorescence time courses were then synchronized withstimulus information, and visually evoked responses were computed aschanges in fluorescence relative to the baseline fluorescence. Peak ΔF/Fresponses for field ROIs and dendritic spines ROIs were computed usingthe Fourier analysis to calculate mean and modulation amplitudes foreach stimulus presentation, which were summed together.

Example 6C: Mouse Neuronal Culture Analysis

Primary Hippocampal Neuron Cultures

Primary hippocampal neuron cultures were prepared from embryonic mice(E16) as described previously (Woitecki et al., 2016, J. Neurosci.,36:2561-70). Hippocampi were rinsed 3-5 times in Hank's Balanced SaltSolution (HBSS, Life technologies) and digested with trypsin (25 mg/ml,Life Technologies) for 20 min at 37° C. followed by DNase I (1 mg/ml;Roche). Subsequently, the tissue was dissociated using cannulas (threetimes 0.9 mm×40 mm; three times 0.45 mm×23 mm) and the solution waspassed through a Nylon cell strainer (100 μm; BD Biosciences). The meshwas rinsed with 4-10 ml basal medium eagle (BME, Life technologies)supplemented with 0.5% glucose (Sigma-Aldrich), 10% fetal calf serum(FCS), 2% B-27, and 0.5 mM L-glutamine (all Life Technologies) tocollect all cells. After counting, the cells were plated on cover slipsin a 24-well cell culture plate at a density of 70,000 cells per 24-welland cultured in a humidified incubator at 37° C. and 5% CO₂.

Viral Vector Production

Recombinant AAV2/1 genomes were generated by large scale tripletransfection of HEK293 cells as described previously (Marvin et al.,2013, Nat. Methods, 10:162-70). The adeno-associated virus (AAV) plasmidcoding for SF-iGluSnFR.S72A or SF-iGluSnFR.A184V, helper plasmidsencoding rep and cap genes (pRV1 and pH21), and adenoviral helper pFΔ6(Stratagene) were transfected using the calcium phosphate transfectionmethod. Cells were harvested ˜72 h after transfection. To purify thevirus, cell pellets were lysed in the presence of 0.5% sodiumdeoxycholate (Sigma) and 50 units/ml Benzonase endonuclease (Sigma).rAAV viral particles were purified from the cell lysate by HiTrapheparin HP column purification (GE Healthcare) and then concentratedusing Amicon Ultra Centrifugal Filters (Millipore) until a final stockvolume of 500 μl was reached.

Viral Transduction and Image Acquisition

Primary hippocampal neurons were transduced withAAV2/1.hSynapsin1.SF-iGluSnFR.S72A or withAAV2/1.hSynapsin1.SF-iGluSnFR.A184V on DIV4 and imaged on DIV13. A lowamplitude field stimulation (1 msec, 20 mA, platinum bar electrodes) wasapplied to recruit a small fraction (˜20%) of neurons. Images wereacquired with an EM-CCD camera (frame time 5-50 msec) and a stabilizedLED light source of cultures visualized through a coverslip with high NAobjective. All experiments were performed in Tyrode's solution (1ml/min) at RT. Low and high affinity versions of SF-iGluSnFR wereexpressed in a comparable manner.

Glutamate Release Site Localization

Primary hippocampal neurons were transduced with rAAV-SF-iGluSnFR. S72Aor with rAAV-SF-iGluSnFR.A184V on DIV3-5 and used for experiments onDIV13-18. A low amplitude electrical field stimulation (1 msec., 20 mA,platinum bar electrodes) was applied to activate a small fraction (˜20%)of neurons only. Per experiment, stimuli were applied 16-25 times at aninter-stimulus interval of 20-60 sec. Images were acquired with anEM-CCD camera (Hamamatsu ImagEM X1, 8 ms exposure, 125 Hz acquisitionrate) attached to an inverted microscope (Nikon T1 Eclipse) using atriggered, stabilized LED light source (Cairn OptoLED with 470 nmexcitation wavelength, 470/40 emission filter and 525/50 excitationfilter). Cells were imaged through a coverslip with a high NA objective(Zeiss, 63×, 1.4 NA, water). All experiments were performed in saline (1ml/min, as described above) at room temperature.

In each experiment, 30 images were acquired per stimulation trial (20before and 10 after stimulation). Each of the 30 images was registeredwith StackReg Plugin in ImageJ to the first image. The image series wasthen normalized to the average of 5 frames before stimulation todistinguish responding sites (>1) and non-responding structures (˜1).For selection of responding sites to be included in the analysis, 10normalized images subsequent to the stimulus in the first trial wereaveraged. All spots of increased fluorescence (FIG. 64c,d ) that reachedat least 50% of the ΔF/F value of the brightest spot in the image weredefined as responding sites and used for further analysis. The spatialextent of glutamate release sites was quantified by extracting abrightness profile based on a line (length: 12-30 pixels, width: 3pixels) drawn along the underlying neurite. These profiles werecalculated for each stimulation trial and each responding site in anexperiment and fitted by Gaussians with Igor Pro 6.3 (Wavemetrics).

In each experiment (n=6 and 8 for S72A and A184V, respectively, eachconsisting of 16-25 trials) the mean deviation of the center (X₀position), the average width and the average amplitude of the fittedGaussians were calculated per response site and averaged across allexperiments and statistically compared by an unpaired Mann-Whitney test,n=28 and 53 for S72A and A184V, respectively).

Example 6D: Cerebellar Parallel Fiber Analysis

Stereotaxic Injections.

To fluorescently label boutons of parallel fibers, stereotaxicinjections of viral vectors expressing SF-iGluSnFR or GCaMP6f intocerebellar vermis were performed. The following vectors were used:AAV-DJ.hSynapsin.SF-iGluSnFR (1.9×10¹³ GC/ml),AAV2/1.hSynapsin.SF-iGluSnFR.S72A (2.6×10¹³ GC/ml),AAV-DJ.CAGFLEX.SF-iGluSnFR.S72A (6.3×10¹² GC/ml) orAAV-DJ.hSynapsin.GCaMP6f (1.2×10¹³ GC/ml). Mice between 30 and 60 daysold were deeply anesthetized before surgery with a mixture of hypnotic(ketamine 1.5%, Mérial) and analgesic (xylazine 0.05%, Bayer)anesthetics mixed in NaCl and injected in the peritoneum. A localanesthetic (xylocaine 2% gel, Newpharma) was applied on top of thelocation of the cranial incision. The anesthetized mouse was then placedon a stereotaxic frame adaptor comprising adjustable ear bars and toothholder. The skull was then perforated at the injection site with asurgical drill. The vermis was identified using the Paxinos and Franklinmouse brain atlas. The injection of viral constructs in the vermis (100nl; 6.5 mm caudal to bregma, lateral 0.2 mm, ventral 3.6 mm and 3.4 mm)was performed by slow infusion (100 nl/min) with steel needles (26G×50mm and 36G×70 mm, Phymep) connected to a pump via a catheter and aHamilton syringe. Injected mice were then kept 2 to 4 weeks to allowtransgene expression.

Slice Preparation

All protocols were approved by the ethics committee CEEA-Paris1.Cerebellar acute slices were prepared from adult CB6F1 mice (F1 cross ofBalbC and C57Bl/6J) or Gabra6 mice (B6;129P2-Gabra6^(tm2(cre)Wwis)/Mmucd) of postnatal day 41 to 123. The micewere killed by rapid decapitation, after which the brains were quicklyremoved and placed in an ice-cold solution containing (in mM): 2.5 KCl,0.5 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, 24 NaHCO₃, 25 glucose, 230 sucrose,and 0.5 ascorbic acid bubbled with 95% O₂ and 5% CO₂. Coronal sliceswere cut from the dissected cerebellar vermis using a vibratome (LeicaVT1200S). After preparation, the slices were incubated at 32° C. for 30minutes in the following solution (in mM): 85 NaCl, 2.5 KCl, 0.5 CaCl₂,4 MgCl₂, 1.25 NaH₂PO₄, 24 NaHCO₃, 25 glucose, 75 sucrose and 0.5ascorbic acid. Slices were then transferred to an external recordingsolution containing (in mM): 125 NaCl, 2.5 KCl, 1.5 CaCl₂, 1.5 MgCl₂,1.25 NaH₂PO₄, 25 NaHCO₃, 25 glucose and 0.5 ascorbic acid, andmaintained at room temperature for up to 6 hours. All slice recordingswere performed at 36-38° C.

Transmitted Light and Fluorescence Imaging

Parallel fiber and boutons expressing SF-iGluSnFR or GCaMP6f wereidentified using an Ultima two-photon scanning scanhead (Bruker NanoSurfaces Division, Middleton, Wis., USA) that was mounted on an OlympusBX61W1 microscope, equipped with a water-immersion objective (60×, 1.1NA, Olympus Optical, Tokyo, Japan) and infrared Dodt-gradient contrast.Two-photon excitation was performed with a pulsed Ti: Sapphire laser(DeepSee, Spectra-Physics, France) tuned to 920 nm for imagingmorphology, glutamate and Ca²⁺ fluorescence detection.

Boutons from parallel fibers were identified by increase fluorescence asresponse to 100 or 300 Hz trains. The probe response was evoked with 60μs voltage pulses 5-15 V above threshold (Digitimer Ltd, LetchworthGarden City, UK) using a patch pipette (typically with a tip resistanceof 4-6 MΩ) filled with ACSF and placed in the molecular layer adjacentto labelled parallel fibers. Activation of boutons was routinelyconfirmed by verifying increase in fluorescence in response to 100 or300 Hz trains of stimulation. Line-scan imaging through boutons wasperformed at dwell time of 0.8 μsec per pixel, for 300 to 800 msec.Individual traces were background subtracted and averaged with nosmoothing or filtration for single events for SF-iGluSnFR, or backgroundsubtracted and averaged with smoothing for GCaMP6f, 20 Hz and 100 Hztrains. SNR was calculated from the peak of the fit to the fluorescentevents divided by the average SD of a 20 msec baseline window. Data wereanalyzed and presented using custom-written macros in Igor Pro.

Example 6E: Fast Imaging of SF-Venus-iGluSnFR

Primary Rat Hippocampal Neuron Cultures

A mixed cell culture (neurons and glia) was prepared from Sprague-Dawleyrat pups (Charles River Laboratories). Briefly, P0 pups weredecapitated, and the brains were dissected into ice-cold neuraldissection solution (NDS, 10 mM HEPES (Sigma) in HBSS (Invitrogen), pH7.4). Hippocampi were dissected and cut into small pieces to facilitateenzyme digestion. Hippocampi pieces were transferred using a large borepipette into a 15 ml conical tube and incubated with enzyme digestsolution (Papain, Worthington Biologicals) at 37° C. for 30 min. After30 min., the enzyme solution was removed, and Plating Media (MEM mediacontaining 10% FBS) was added and tissue pieces were trituratedresulting in mostly single cells. The cell suspension was filtered usinga 45 um filter. The filtered cell suspension was centrifuged, and theresulting cell pellet was re-suspended with Plating Media and counted.

For electroporation, 1 μg of DNA was mixed with 1×10⁶ cells using theAmaxa Nucleofector II instrument. Cells were plated onto coverslipscoated with Poly-D-Lysine (Sigma) and kept at 37° C., 5% CO₂ in PM for˜24 hours and then in NbActiv4 (BrainBits) was added for the durationwith medium exchanges every 4 days.

Glutamate Uncaging and Imaging

Rat hippocampal culture was imaged on DIV19 at room temperature in HEPESbuffered Tyrode's solution (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10mM HEPES, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4).

Excitation was with a 1030 nm, 5 MHz, 190 fsec laser (Menlo Systems,model: Bluecut). Average power was 39 mW at the sample. Fluorescencecollected at 560/80 nm with a Hamamatsu MPPC detector. The field of viewis a 256 um diameter circle, 1280 pixels across. The bath containedHEPES buffered Tyrode's solution plus 10 μM NBQX and 150 μMRuBi-Glutamate (Tocris). Glutamate uncaging was performed with 420 nmfiber-coupled LEDs (Thorlabs M420F2). The tips of the fibers were imagedonto the sample plane through the same objective used for activityimaging.

Example 6F: Summary of Results

The intensity-based glutamate-sensing fluorescent reporter (iGluSnFR)(Marvin et al., 2013, Nat. Methods, 10:162-70) has become an invaluabletool for studying glutamate dynamics in diverse systems, includingretina (Park et al., 2014, J. Neurosci., 34:3976-81; Borghuis et al.,2013, J. Neurosci., 33:10972-85), mouse olfactory bulb (Brunert et al.,2016, J. Neurosci., 36:6820-35) and cat visual cortex (O'Herron et al.,2016, Nature, 534:378-82). Beyond specific circuits, iGluSnFR alsoallows mesoscale “functional connectomic” mapping (Xie et al., 2016, J.Neurosci., 36:1261-72) and mechanistic studies of Huntington's disease(Jiang et al., 2016, J. Neurosci., 36:3453-70), synaptic spillover (Rosaet al., 2015, eLife, 4:728), cortical spreading depression (Enger etal., 2015, Cerebral Cortex, 25:4469-76) and exocytotic vesicle fusion(Bao et al., 2016, Nat. Struct. Biol., 23:67-73). However, iGluSnFR isinsufficient for some applications due to poor expression (in some brainregions), and kinetics that do not match the time courses of someobservations. Here, we describe variants that are functionally brighter(due to increased expression on cell membrane), have tighter or weakeraffinity (resulting from slower or faster off-rates), and fluoresceblue, green, or yellow.

Replacement of circularly permuted eGFP with circularly permuted“superfolder” GFP (Pedelacq et al., 2006, Nat. Biotech., 24:79-88)(SF-iGluSnFR) yielded 5-fold higher soluble-protein expression levels inbacteria (0.5 μmol/1 L growth vs. 0.1 μmol/1 L). Circular dichroismindicates an increase in melting temperature transition (T_(m)) of ˜5°C. (FIG. 57). The 2-photon cross-section and excitation, emission, andabsorption spectra of SF-iGluSnFR are similar to the original (FIG.58a-d ). Head-to-head comparison of SF-iGluSnFR with original iGluSnFRin mouse somatosensory cortex shows substantially more robust expressionby the former (FIG. 59a,b ). Under typical imaging conditions (<20 mW,130-nanosecond dwell time per pixel), SF-iGluSnFR is bright enough forrepeated imaging, while original iGluSnFR is too dim (FIG. 59c,d ).While a faster 2-photon in vivo photobleaching rate was observed forSF-iGluSnFR in somatosensory cortex (FIG. 59e ), partially-bleachedSF-iGluSnFR was still brighter than iGluSnFR. Thus, SF-iGluSnFR willhave superior expression in vivo, where the quantity of deliverable DNAcan be limiting.

While the affinity of membrane-displayed iGluSnFR (4 μM) is adequate forsome in vivo applications, tighter variants are needed for circumstancesof limiting glutamate concentrations, such as at sparsely-firingsynapses. Additionally, measuring glutamate release events with rasterscanning microscopes requires variants with slower off-rates so that thedecay time from glutamate binding is long enough to be sufficientlysampled at the operating frame rate for most experiments (typically <100Hz). Replacement of eGFP with superfolder GFP increases the in vitroaffinity of soluble SF-iGluSnFR two-fold compared to original iGluSnFR(40 μM vs. 80 FIG. 60a ). To further modulate affinity, theconformational coupling between the open-closed equilibrium of bacterialperiplasmic binding proteins (PBPs, e.g. the glutamate-binding proteinin iGluSnFR) and their ligand-binding affinity (Marvin et al., 2001,Nat. Struct. Biol., 8:795-8) was exploited. Briefly, mutation ofresidues in the “hinge” of PBPs can allosterically alter affinity,without compromising the stereochemical integrity of the ligand-bindingsite. In a bacterial lysate assay, an A184X library of the iGluSnFRglutamate-binding domain (mutated to valine in the original iGluSnFR)was screened. Reversion to alanine or other small amino acids tightenedaffinity, while larger side chains weakened affinity (FIG. 61).

A184S was introduced into SF-iGluSnFR to generate a tighter variant.(Reversion A184A had a low ΔF/F.) Affinities of purified soluble proteinwere 7 μM and 40 μM for the A184S and A184V (unmutated from iGluSnFR)SF-iGluSnFR variants, respectively (FIG. 60a ). The tighter affinity ofthe A184S variant arises from a slower off-rate (FIG. 60b ). Theaffinity variants were re-cloned into an AAV vector containing an IgGsecretion signal and a PDGFR transmembrane domain. Viral expression oncultured rat hippocampal neurons (AAV2/1.hSynapsin1.SF-iGluSnFR) yieldsglutamate affinities about an order of magnitude tighter than thesoluble form (0.7 μM and 2 μM for A184S and A184V, respectively; FIG.62). A similar increase in affinity upon membrane tethering was seenwith the original sensor (Marvin et al., 2013, Nat. Methods, 10:162-70).Whole-field stimulation (50 Hz) of these cultures shows that theirrelative half-times of fluorescence decay parallel their in vitrokinetics, with all variants having faster decay than GCaMP6f (FIG. 63).

In vivo, the tighter/slower SF-iGluSnFR.A184S variant shows improveddetection of stimulus-evoked glutamate release in the ferret visualcortex in response to presented drifting gratings (FIG. 53a,b ). Peakamplitudes reached 30% ΔF/F for SF-iGluSnFR.A184S but only 5% ΔF/F forSF-iGluSnFR.A184V when imaged at 30 Hz. Greater ΔF/F ofSF-iGluSnFR.A184S allows extraction of robust orientation tuning curvescompared to SF-iGluSnFR.A184V. Enhanced sensitivity of the A184S variantalso allowed orientation-selective responses to be resolved inindividual dendritic spines (FIG. 53c,d ). Synaptic glutamate release asmeasured with SF-iGluSnFR.A184S was not only strongly selective forvisual stimuli, but response amplitudes across individual trials wereconsistently greater than the A184V variant when examining allstimulus-evoked responses (A184S median ΔF/F=16%, n=72 spines; A184Vmedian ΔF/F=9%, n=22 spines; p=2e-115, Wilcoxon rank-sum test) or onlypreferred stimuli (A184S median ΔF/F=27%, n=72 spines; A184V medianΔF/F=14%, n=22 spines; p=9e-23, Wilcoxon rank-sum test) (FIG. 64).

While slow off-rate variants of SF-iGluSnFR are better for detectingindividual synaptic events by temporal summation of fluorescence, fasteroff-rate variants are needed for temporal resolution spiking dynamicsand at large synapses where glutamate clearance is limiting. A weakervariant of SF-iGluSnFR (S72A) was made by removing a hydrogen bondbetween the protein and glutamate. Soluble SF-iGluSnFR.S72A has 200 μMaffinity for glutamate (FIG. 60a ), arising from a combination of bothslower on-rate and faster off-rate (FIG. 60b ). In neuronal culture,S72A has an affinity of 35 μM, an order of magnitude weaker than itsparent, A184V (FIG. 62).

In rat neuronal culture, without buffer perfusion, fluorescence of theculture (not localized to specific structures) returns to baselinewithin 100 msec. of a single electrical stimulation for S72A, fasterthan A184V, A184S, or GCaMP6f (FIG. 63). In mouse neuronal culture (FIG.65), the substantially faster off-rate of S72A provides enhancedtemporal resolution of paired (20 Hz) electrical stimuli over the A184Vvariant (FIG. 54a,b ), making it useful for assessing short-termsynaptic plasticity. A train of 6 electrical pulses (20 Hz) in 1 mMextracellular Ca²⁺ can be resolved as equal, individual release eventsby observation with S72A, while A184V yields an integrated signal (FIG.54c,d ). In 3.5 mM extracellular Ca²⁺, vesicles are released with higherprobability during the initial stimulation (Dodge et al., 1967, J.Physiol., 193:419-32). This can be observed by S72A, as reported by areduction in fluorescence response as the train of field pulsesprogresses (FIG. 54c ), while these differences are obscured by theslower decay of A184V (FIG. 54d ). Thus, while S72A has a lower ΔF/F inresponse to the same amount of glutamate being released (due to weakeraffinity), its faster kinetics provides enhanced temporal resolution ofsynaptic activity. Similarly, S72A provides enhanced spatial resolutionof glutamate release over A184V (FIG. 66).

With fast rise and decay times, it was examined whether SF-iGluSnFRcould be used as an alternative to GCaMP6f for monitoring neuronalactivity in mouse cerebellar brain slice. Single cerebellar granule cellbouton responses to single action potentials (APs) could indeed beresolved using fast linescan detection (<1 ms per line; FIG. 55a ), andwere much faster than GCaMP6f rise and decay times at both 2 mM and 1.5mM extracellular calcium. The S72A variant had by far the fastestresponse (S72 half decay 7.9±1.0 ms, A184V 28.1±1.6 ms, GCaMP6f 1.5 mM[Ca²⁺]_(e) 37.9±3.9 ms, GCaMP6f 1.5 mM [Ca²⁺]_(e) 108.6±8.8 ms). Thesignal-to-noise-ratios (SNRs) were best for A184V, but even S72Aproduced better SNRs than GCaMP6f under physiological extracellularcalcium concentrations (1.5 mM). The superior SNR of A184V showedputative single vesicle release events in single trials (FIG. 55b ).However, if many bouton responses are pooled and averaged for eachtrial, single spike detection at 20 Hz is feasible (see average trace,FIG. 55b ). For 20 Hz stimuli, both the A184V and S72A variants producedlittle accumulation of bouton fluorescence after 10 stimuli as comparedto GCaMP6f (FIG. 55c ), similar to the dendritic responses in culture(FIG. 54). For 100 Hz train stimuli, discrete release events could bedetected, in contrast to GCaMP6f (FIG. 55d ). Note the poor temporalprecision of the train response, in contrast to A184V and S72A. Thusboth A184V and S72A enable a larger dynamic range of reported firingfrequencies, with S72A providing the largest range due to its lowaffinity. Moreover, the fast kinetics of SF-iGluSnFR.A184V andSF-iGluSnFR.S72A could be used for a more reliable estimate of spiketimes (versus GCaMP6f), and are much better suited to high-frequencyspike detection (>100 Hz) which is necessary for the high instantaneousfiring rates of cerebellar granule cells (van Beugen et al., 2013,Frontiers in Neural Circuits, 7:95).

Introduction of chromophore mutations from GFP variants Azurite (Mena etal., 2006, Nat. Biotech., 24:1569-71) or Venus (Nagai et al., 2002, Nat.Biotech., 20:87-90) to SF-iGluSnFR led to functional blue and yellowversions, respectively. The former required re-optimization of theresidues that link the FP with the glutamate-binding protein. The latterwas a straightforward modular replacement. (Annotated amino acidsequences are given in FIG. 67). SF-Azurite-iGluSnFR has significantlylower ΔF/F (FIG. 68), perhaps a result of intrinsic differences inchromophore structure. SF-Venus-iGluSnFR has similar affinity andmaximum fluorescence response to glutamate as SF-iGluSnFR, but withred-shifted excitation and emission spectra (FIG. 69). Importantly, its2-photon excitation spectrum is sufficiently red-shifted to allow strongexcitation at 1030 nm (FIG. 69), compatible with relatively inexpensive,powerful femtosecond fiber lasers (Tang et al., 2009, J. Biomed. Optics,14:030508). These powerful lasers enable simultaneous excitation of manyfoci, enabling very fast (1016 Hz) large-area imaging by recordingprojections of the sample and computationally reconstructing images(Kazemipour, et al., 2018). In neuronal culture, two near-simultaneouspulses of glutamate uncaging can be resolved with both high spatial andtemporal resolution by measuring fluorescence changes in a neuronexpressing SF-Venus-iGluSnFR.A184V (FIG. 56).

The iGluSnFR variants described here increase the power of geneticallyencoded glutamate imaging. Affinity variants with altered kineticsbroaden the range of observable glutamate release events. Chromaticmutants allow fast imaging with cheap lasers, and potential utility inmulti-color imaging. Improved membrane targeting and photostability willbe valuable in all applications.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A recombinant peptide biosensor comprising ananalyte-binding framework portion and a signaling portion, wherein thesignaling portion is present within the framework portion at a site oramino acid position that undergoes a conformational change uponinteraction of the framework portion with a defined, specific, orselected analyte, wherein the recombinant peptide biosensor comprises aamino acid sequence having at least 85% sequence identity to SEQ ID NO:184.
 2. The recombinant peptide biosensor of claim 1, wherein thesignaling portion is allosterically regulated by the framework portionsuch that signaling from the signaling portion is altered uponinteraction of the framework portion with the analyte.
 3. Therecombinant peptide biosensor of claim 1, wherein signaling by thesignaling portion detectably increases upon interaction of the frameworkportion with the analyte.
 4. The recombinant peptide biosensor of claim1, wherein signaling by the signaling portion detectably decreases uponinteraction of the framework portion with the analyte.
 5. Therecombinant peptide biosensor of claim 1, wherein signaling by thesignaling portion is proportional to the level of interaction betweenthe framework portion and the analyte.
 6. The recombinant peptidebiosensor of claim 1, wherein the framework portion has a firststructure in the absence of an analyte and a second structure, that isdetectably distinct from the first structure, in the presence of theanalyte.
 7. The recombinant peptide biosensor of claim 6, wherein theconformational change between the first structure and the secondstructure allosterically regulates the signaling portion.
 8. Therecombinant peptide biosensor of claim 1, wherein the framework portionis a periplasmic binding protein (PBP) or a variant of a PBP.
 9. Therecombinant peptide biosensor of claim 1, wherein the signaling portionis a circularly permuted super fluorescent (SF) protein.
 10. Therecombinant peptide biosensor of claim 9, wherein the SF protein isselected from the group consisting of a green fluorescent protein, ayellow fluorescent protein, a red fluorescent protein, and a bluefluorescent protein.
 11. The recombinant peptide biosensor of claim 1,wherein the analyte-binding framework portion binds specifically togamma aminobutyric acid (GABA).
 12. A recombinant peptide biosensorcomprising an amino acid sequence having at least 90% sequence identityto SEQ ID NO:184, wherein the recombinant peptide biosensor bindsspecifically to GABA.
 13. The recombinant peptide biosensor of claim 12having SEQ ID NO:184 and comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to GABA.
 14. The recombinant peptide biosensor of claim 12,comprising a recombinant peptide biosensor having SEQ ID NO:184.
 15. Anucleic acid encoding the recombinant peptide biosensor of claim
 1. 16.A vector comprising the nucleic acid of claim
 15. 17. A cell comprisingthe nucleic acid of claim
 15. 18. A cell comprising the vector of claim16.
 19. A kit comprising the recombinant peptide biosensor of claim 1,the nucleic acid of claim 15, the vector of claim 16, the cell of claim17, and/or the cell of claim
 18. 20. A method for detecting GABA, themethod comprising detecting a level of fluorescence emitted by arecombinant peptide biosensor, the peptide biosensor having the aminoacid sequence shown in SEQ ID NO:184, and correlating the level offluorescence with the presence of GABA.
 21. The method of claim 20,wherein the recombinant peptide biosensor is expressed from a nucleicacid.
 22. The method of claim 20, comprising contacting the recombinantpeptide biosensor with a sample comprising GABA.
 23. The method of claim22, comprising correlating the level of fluorescence with aconcentration of GABA.
 24. The method of claim 23, comprising comparingthe level of fluorescence with a level of fluorescence emitted by therecombinant peptide biosensor in the presence of a sample comprising aknown concentration or range of concentrations of GABA.
 25. The methodof claim 24, wherein the method is performed in vitro.
 26. A method fordetecting a defined, selected, or specific analyte, the methodcomprising detecting a level of fluorescence emitted by a recombinantpeptide biosensor of claim 1 in the presence of said analyte; andcorrelating the level of fluorescence with the presence of a defined,selected, or specific analyte.
 27. The method of claim 26, wherein therecombinant peptide biosensor is expressed from a nucleic acid.
 28. Themethod of claim 26, comprising contacting the recombinant peptidebiosensor with a sample comprising the analyte.
 29. The method of claim28, comprising correlating the level of fluorescence with aconcentration of the analyte.
 30. The method of claim 29, comprisingcomparing the level of fluorescence with a level of fluorescence emittedby the recombinant peptide biosensor in the presence of a samplecomprising a known concentration or range of concentrations of theanalyte.
 31. The method of claim 30, wherein the method is performed invitro.
 32. The method of claim 30, wherein the analyte is GABA.